Method to assess prognosis and to predict therapeutic success in cancer by determining hormone receptor expression levels

ABSTRACT

The present invention is related to a method of classifying a sample of a patient who suffers from or being at risk of developing cancer, said method comprising the steps of determining in said sample from said patient, on a non protein basis, the expression level of at least one gene encoding for a hormone receptor selected from the group comprising estrogen receptor, progesterone receptor and/or androgen receptor in said sample; comparing the one or more expression level(s) determined with one or more expression level(s) of one or more reference genes, and classifying the sample of said patient from the outcome of the comparison into one of at least two classifications.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to International Application No.PCT/EP2010/055745 filed on 28 Apr. 2010, which, in turn, claims priorityfrom Patent Application No. EP 09159005.9, filed on 29 Apr. 2009, eachof which is incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a method to assess the prognosis ofcancer and to predict therapeutic outcome in cancer treatment. Theinvention has been tested to be useful for different cancer diseasessuch as but not limited to lung, ovarian, breast and prostate cancer.

BACKGROUND OF THE INVENTION

Cancer is a class of diseases in which a group of cells displayuncontrolled growth (division beyond the normal limits), invasion(intrusion on and destruction of adjacent tissues), and sometimesmetastasis (spread to other locations in the body via lymph or blood).

Lung cancer is a cancerous disease of uncontrolled cell growth intissues of the lung. This growth may lead to metastasis, which is theinfiltration and invasion of adjacent tissue and infiltration beyond thelungs. The vast majority of primary lung cancers are carcinomas of thelung, derived from epithelial cells.

Response to chemotherapy in lung cancer is comparatively low with about10%-30% of patients having benefit from treatment, while having seriousside effects and being costly for the national health systems. Despiteresponsiveness towards chemotherapy, the survival of lung cancerpatients is still very poor. Lung cancer is the most lethal cancer inthe world with estimated 215,000 new cases and 162,000 deaths per yearin the US (Jemal, et al. 2008; 5 year overall survival: Stage I 50%,Stage IV 3%) in part due to the fact that most cases are detected in thelater stages.

It is a well-established fact, that systemic treatment after surgeryreduces the risk of disease relapse and death in patients with primaryoperable cancer. However, there still are a great number of patients whodo not benefit from systemic therapy.

Prognostic factors in lung cancer include presence or absence ofpulmonary symptoms, tumor size, cell type (histology), degree of spread(stage) and metastases to multiple lymph nodes, and vascular invasion.

There are only few data addressing the molecular prediction of responseto therapy in lung cancer. Endocrine therapies have not been consideredfor early treatment of lung cancer so far mostly for several reasons:lung cancer is not a gynecologic tumor site, estrogens do not play amajor role in lung development, significant (initial) response tochemotherapy and lack of stratification marker for endocrine therapies.This is in part due to the fact that the determination of hormonereceptors and particularly ESR1 by immunohistochemistry failed to haveprognostic value for lung cancer, while ESR2 determined on protein levelmay have some relevance in the comparatively small group of EGFR mutatedtumors (Nose N, Sugio K, Oyama T, Nozoe T, Uramoto H, Iwata T, OnitsukaT, Yasumoto K.: Association between estrogen receptor-beta expressionand epidermal growth factor receptor mutation in the postoperativeprognosis of adenocarcinoma of the lung. J Clin Oncol. 2009 Jan. 20;27(3):411-7. Epub 2008 Dec. 8.).

This all is in sharp contrast to breast cancer, where the role of ESR1mRNA and ER protein expression is well established as a stratificationmarker for endocrine treatment options.

In sharp contrast to, e.g., Nose, et al. (2009), who have not found anyprognostic role of ER protein expression in 447 resected primary lungadenocarcinoma, we have surprisingly found significant results obtainedby using the RNA extraction and target gene determination approachdescribed below.

Markers predicting tumor response can function as sensitive short-termsurrogates of long term outcome. Response to primary chemotherapy is anexcellent experimental model to study the efficacy of anticancer therapyin a relatively short period of time. Moreover, the molecular analysisof pre- and post-chemotherapy tumor specimen enables the identificationof chemotherapy resistant tumor cell subpopulation and thereby leads toadapted treatment options. However the identification of relevantresistance mechanisms in such settings and development of tests thatcould be used to detect these underlying resistance mechanisms forpatient selection before therapy in clinical routine tissue have notsucceeded so far. The use of such markers can make therapeuticstrategies more effective for the individual patient and will allowchanging regimen early in the case of non-responding tumors. Moreover,the identification of such markers has the potential to identify newdrug targets and develop new and more effective treatments.

Lung cancer is commonly treated by chemotherapy, radiotherapy, orsurgery with adjuvant chemotherapy. While hormonal therapies arecommonly used in the treatment of endocrine organ-associatedmalignancies such as breast and prostate cancer, at present they are notindicated for lung cancer cases.

Ovarian cancer is the most lethal gynecologic cancer with 20,000 newcases per year and 15,000 deaths per year in the US (5 year overallsurvival: Stage I 80%, Stage IV 20%) in part due to the fact that mostcases are detected in the late stages III and IV). Chemotherapy isstandard of care for early and advanced ovarian cancer, while endocrinetherapy is given only after failure of chemotherapy regimens. Responseto chemotherapy is comparatively low with about 10%-30% of patientshaving benefit from treatment, while having serious side effects andbeing costly for the national health systems. Despite prominentresponses towards chemotherapy, the survival of particularly ovariancancer patients is still very poor. To date there are no reliableresponse markers to predict response to chemotherapy or endocrinetherapy in ovarian cancer based on immunohistochemistry, FISH orexpression profiling analysis. However, there are only few dataaddressing the molecular prediction of response to therapy in ovariancancer.

Prostate cancer is the most frequent male cancer with approximately190,000 new cases per year in the United States. However, in contrast tothe situation in lung and ovarian cancer, most tumors are identified inan early and yet good prognostic stage. Compared to the high incidencerate the annual death rate is therefore comparably low withapproximately 30,000 deaths. For most prostate cancer patients “watchfulwaiting”, i.e., sparing patients surgery, radiation and systemictreatment would be the most appropriate way to treat prostate cancerpatients, as the individual risk of distant metastasis and death is verylow (=“progression risk”). This is of particular importance given thehigher age and comorbidities of prostate cancer patients. Moreover,therapeutic approaches to treat prostate cancer all bear a high risk ofdeveloping significant and persistent side effects, such as incontinenceand impotence in about 80% of the cases. However, there are no reliablemarkers that might be useful to reliably identify patients of lowprogression risk and be useful for tailored treatment approaches. Aparticular problem is the high heterogeneity and dispersed growth ofprostate cancer. Biopsying and subsequent tissue analysis is thereforeonly of limited efficacy and prognostic value.

Despite state of the art chemo- and endocrine therapy, more than 15% ofall breast cancer patients metastasize early and die within the firstthree to five years after initial surgery. Multiple studies havedemonstrated that adjuvant therapy for early-stage breast cancerproduces a 23% or greater improvement in disease-free survival and a 15%or greater increase in overall survival rates. However, 30% of breastcancer patients suffer from recurring disease even after harshchemotherapeutic and endocrine treatment and 15% of the patients diewithin four years after primary surgery.

In general, all patients of a given cohort do receive the sametreatment, even though many will fail in treatment success. Markerspredicting tumor response can function as sensitive short-termsurrogates of long-term outcome. The use of such markers can makechemotherapy more effective for the individual patient and will allowchanging regimen early in the case of non-responding tumors.

Although much effort has been devoted in developing an optimal clinicaltreatment course for individual patients with cancer, very littleprogress has been made in predicting the individual's response to acertain treatment. Currently, the probability of response of patients toa certain cancer treatment is usually determined by measuring the statusof a marker on protein-level by immunohistochemistry (IHC). Assays basedon protein-level measurements exhibit only limited quantitativeperformance and comparatively high inter- and intra-assay variabilities.Especially immunohistochemistry often yields different results indifferent laboratories. IHC assays have the added drawback that theyoften need to be evaluated by trained pathologists or other personnel,thus adding a subjective component to the determination of assayresults.

Other approaches, as FISH (Fluorescence In Situ Hybridization) orexpression profiling analysis, suffer of drawbacks as low sensitivity,restriction of sample preparation and restricted multiplexingcapabilities.

Chemotherapy is standard of care for early and advanced lung cancer,while endocrine therapies have not been tested in this cancerindication. To date there are no reliable response markers to predictresponse to chemotherapy or endocrine therapy in lung cancer based onimmunohistochem-istry, FISH or expression profiling analysis. So, it isyet difficult to determine those patients suffering of lung cancer whowill respond to a certain therapy.

Similarly, in ovarian cancer endocrine therapies have not been tested inearly treatment stages. Lack of reliable response markers and failure ofimmunohistochemical methods to determine the prognostic value of hormonereceptors has corrupted these developments. In contrast in breast andprostate cancer the endocrine treatment options are standard of care asbeing one of the most effective treatment options. Here, the reasons forfailure of endocrine treatment, is still not well understood.

The present invention surprisingly opens a new approach to diagnosticassessment of cancer and also suggests the possibility of endocrinetherapy for cancer patients. Moreover it enables a new kind of cancertumor classification into the principle underlying biological activitiesand therefore a general risk categorization resembling to some extentthe current situation in breast cancer.

BASIS OF THE INVENTION

In several cancer diseases, the determination of hormone receptors byimmunohistochemistry so far has failed to have prognostic value.Surprisingly, even in cancers that have been recalcitrant tohormone-based therapies thus far, the inventor of the present inventionhas found methods by which the determination of hormone receptor statuscan have prognostic significance.

The significance of hormone receptor status in cancers of the femalebreast or reproductive organs, including uterus, ovaries, cervix,fallopian tubes, vulva, vagina, prostate and testes, is well known. Insome of these cancers, e.g., breast cancer, the determination of hormonereceptor status is standard medical practice. Lung tissues, however,unlike tissues of the reproductive organs, are not generally known to begrowth regulated by steroid hormones. It is particularly surprising andunexpected that hormone receptor status should have a prognostic valuein such types of cancer.

Moreover, it is new that hormone receptor status of female cancerpatients determined according to said methods should be taken intoaccount, when intending to administrate hormone replacement therapy(HRT) to peri- or postmenopausal women. Here, elevated hormone receptorlevels, such as, e.g., estrogen receptor ESR1, and/or low levels ofsnail factors, such as, e.g., SNAI2, indicate not to administerhormones, as this could force aggressiveness and progression of anotherwise comparably less harmful or low risk tumor.

Determining the expression levels of hormone receptors and counteractingtranscription factors involved in epithelial-mesenchymal-transition(“EMT”), that directly and negatively affect the hormone receptorexpression level, improves said method with regard to higher robustnessand lower technical complexity. By generating a two gene ratio betweenhormone receptors (e.g., ESR1) and EMT-transcription factors (e.g.,SNAIL2), the hormone receptor status becomes more precise and robust.Surprisingly this two gene ratio not only performed in lung cancer, butalso in ovarian, prostate and breast cancer, indicating that the balancebetween hormone receptors and EMT markers is generally critical withregard to survival and response to treatment in cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Kaplan-Meier-Analysis of overall survival (OAS) of patientssuffering from lung cancer based on i ESR1 (ER) determination. Theoverall survival (OAS) is depicted in months. Patients are stratifiedaccording to ESR1 expression above or below the median ESR1 expression.

FIG. 2: Kaplan-Meier-Analysis of overall survival (OAS) of patientssuffering from lung cancer based on i ESR1 (ER) determination. Theoverall survival (OAS) is depicted in months. Patients are stratifiedaccording to ESR1 expression above or below the third quartile of ESR1expression.

FIG. 3: Spearman correlation analysis between affected metastatic sitesand ESR1 mRNA expression level in NSCLC patients. Patients are depictedaccording to ESR1 expression above and below the median (i.e., “1” vs“0” respectively). Metastatic site being affected is depicted as “1” or“0” depending on whether metastatic lesions were found before first linetreatment.

FIG. 4: Spearman correlation analysis between candidate genes (ESR1,SNAI2, CDH1, CDH11). Spearman correlation coefficients and p-values aredepicted.

FIG. 5: Kaplan-Meier-Analysis of Recurrence Free Survival (RFS) ofpatients suffering from ovarian cancer based on combined SNAI2 and ESR1determination. The Recurrence Free Survival (RFS) is depicted in months.Patients are stratified according to SNAI2/ESR1 two gene ratios.

FIG. 6: Kaplan-Meier-Analysis of Recurrence Free Survival (RFS) ofpatients suffering from lung cancer based on combined SNAI2 and ESR1determination. The Recurrence Free Survival (RFS) is depicted in months.Patients are stratified according to SNAI2/ESR1 two gene ratios.

FIG. 7: Kaplan-Meier-Analysis of Recurrence Free Survival (RFS) ofpatients suffering from lung cancer based PGR expression determination.The Recurrence Free Survival (RFS) is depicted in months. Patients arestratified according to PGR expression levels above or below the cut-offvale of 2.28 dividing the cohort in ˜65% Low Risk patients and ˜35% HighRisk patients.

FIGS. 8-16: SEQ ID NOs 1-9, respectively.

DEFINITIONS

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

The term “prediction” as used herein relates to the likelihood that apatient will respond either favorably or unfavorably to a given therapy.Especially, the term “prediction”, as used herein, relates to anindividual assessment of the malignancy of a tumor, or to the expectedsurvival rate (DFS, disease free survival) of a patient, if the tumor istreated with a given therapy. In contrast thereto, the term “prognosis”relates to an individual assessment of the malignancy of a tumor, or tothe expected survival rate (DFS, disease free survival) of a patient, ifthe tumor remains untreated.

The term “predicting an outcome” of a disease, as used herein, is meantto include both a prediction of an outcome of a patient undergoing agiven therapy and a prognosis of a patient who is not treated. The term“predicting an outcome” may, in particular, relate to the risk of apatient suffering an event, such as metastasis or death, preferablywithin a given time frame.

The term “classification of a sample” of a patient, as used herein,relates to the association of said sample with at least one of at leasttwo categories. These categories may be for example “high risk” and “lowrisk”, high, intermediate and low risk, wherein risk is the probabilityof a certain event occurring in a certain time period, e.g., occurrenceof metastasis, disease free survival, and the like. It can further meana category of favorable or unfavorable clinical outcome of disease,responsiveness or non-responsiveness to a given treatment or the like.Classification may be performed by use of an algorithm, in particular adiscriminant function. A simple example of an algorithm isclassification according to a first quantitative parameter, e.g.,expression level of a gene of interest, being above or below a certainthreshold value. Classification of a sample of a patient may be used topredict an outcome of disease. Instead of using the expression level ofa single gene of interest, a combined score of several genes of interestmay be used. Further, additional data may be used in combination withthe first quantitative parameter. Such additional data may be clinicaldata from the patient, such as sex, age, weight of the patient, tumorgrading or stage, and the like.

A “discriminant function” is a function of a set of variables used toclassify an object or event. A discriminant function thus allowsclassification of a patient, sample or event into a category or aplurality of categories according to data or parameters available fromsaid patient, sample or event. Such classification is a standardinstrument of statistical analysis well known to the skilled person. Forexample, a patient may be classified as “high risk” or “low risk”, “highprobability of metastasis” or “low probability of metastasis”, “in needof treatment” or “not in need of treatment” according to data obtainedfrom said patient, sample or event. Classification is not limited to“high vs. low”, but may be performed into a plurality of categories,grading or the like. Examples for discriminant functions which allow aclassification include, but are not limited to discriminant functionsdefined by support vector machines (SVM), k-nearest neighbors (kNN),(naive) Bayes models, or piece-wise defined functions such as, forexample, in subgroup discovery, in decision trees, in logical analysisof data (LAD) and the like.

The term “response marker” relates to a marker which can be used topredict the clinical response of a patient towards a given treatment.Response includes direct observation of tumor shrinkage upon neoadjuvantor palliative treatment as evident by, e.g., CT-Scans and/or serumbiomarkers as well as effects on Disease Free Survival (DFS), OverallSurvival (OAS), Metastasis Specific Survival (MSS), Disease SpecificSurvival and related assessments.

The term “clinical response” of a patient, as used herein, relates tothe effectiveness of a certain therapy in a patient, meaning animprovement in any measure of patient status, including those measuresordinarily used in the art, such as overall survival, progression freesurvival, recurrence-free survival, and distant recurrence-freesurvival. Recurrence-free survival (RFS) refers to the time (in years)from surgery to the first local, regional, or distant recurrence.Distant recurrence-free survival (DFRS) refers to the time (in years)from surgery and/or initial diagnosis to the first anatomically distantrecurrence. The calculation of these measures in practice may vary fromstudy to study depending on the definition of events to be eithercensored or not considered. The term “response marker” relates to amarker which can be used to predict the clinical response of a patienttowards a given treatment.

The term “neoplastic disease” refers to a cancerous tissue this includescarcinomas, e.g., carcinoma in situ, invasive carcinoma, metastaticcarcinoma, and pre-malignant conditions, neomorphic changes independentof their histological origin. The term “adenocarcinoma” refers to amalignant tumor originating in glandular tissue. The terms “cancer” and“cancerous” refer to or describe the physiological condition in mammalsthat is typically characterized by unregulated cell growth. The term“cancer” is not limited to any stage, grade, histomorphological feature,invasiveness, aggressiveness or malignancy of an affected tissue or cellaggregation. In particular stage 0 cancer, stage I cancer, stage IIcancer, stage III cancer, stage IV cancer, grade I cancer, grade IIcancer, grade III cancer, malignant cancer, primary carcinomas, and allother types of cancers, malignancies and transformations speciallyassociated with gynecologic cancer are included. The terms “neoplasticdisease” or “cancer” are not limited to any tissue or cell type theyalso include primary, secondary or metastatic lesions of cancerpatients, and also comprise lymph nodes affected by cancer cells orminimal residual disease cells either locally deposited or freelyfloating throughout the patient's body.

As used herein, the term “lung cancers” refers to cancer or malignancieswhich are diagnosed in the lung and is meant to include all cancers,neoplastic growths and cancerous transformations of lung tissue.Examples of lung cancers include, but are not limited to: small celllung carcinoma (SCLC), and non-small cell lung carcinoma (NSCLC), inparticular squamous cell lung carcinoma, adenocarcinoma,bronchioloalveolar carcinoma, large cell lung carcinoma, and others,such as pleuropulmonary blastoma and carcinoid tumors.

The term “tumor” as used herein, refers to all neoplastic cell growthand proliferation, whether malignant or benign, and all pre-cancerousand cancerous cells and tissues.

The term “neoplastic cells” refer to abnormal cells that grow byincreased cellular proliferation, altered cell division symmetry ordecreased cell death mechanisms more rapidly than normal. As such,neoplastic cells of the invention may be cells of a benign neoplasm ormay be cells of a malignant neoplasm.

Furthermore, the term “characterizing the state” of a neoplastic diseaseor cancer is related to, but not limited to, measurements and assessmentof one or more of the following conditions: Type of tumor,histomorphological appearance, dependence on external signal (e.g.,hormones, growth factors), invasiveness, motility, state by TNMClassification of Malignant Tumors (TNM), a cancer staging systemdeveloped and maintained by the International Union Against Cancer, orsimilar, agressivity, malignancy, metastatic potential, andresponsiveness to a given therapy.

The terms “therapy modality”, “therapy mode”, “regimen”, “chemoregimen”, and “therapy regimen” each refer to a timely sequential orsimultaneous administration of anti-tumor, and/or anti-vascular, and/orimmune stimulating, and/or blood cell proliferative agents, and/orradiation therapy, and/or hyperthermia, and/or hypothermia—any and allapproaches for cancer therapy. The administration of these approachescan be performed in an adjuvant and/or neoadjuvant mode. The compositionof any such “protocol” may vary in the dose of the single agent,timeframe of application and frequency of administration within adefined therapy window. Currently various combinations of various drugsand/or physical methods, and various schedules are under investigation.

The term “endocrine treatment” refers to various treatment modalitiesknown as hormonal therapy or anti-hormonal therapy that produce thedesired therapeutic effect by means of change of hormone/hormones level.The treatment may include administration of hormones or hormone analogs,synthetic hormones or other drugs to the patient, or decreasing thelevel of hormones in the body by using hormone antagonists, hormonereceptor antagonists or hormone ablation therapy either by surgicalresection of ovaries or by chemical suppression of hormone synthesis.Endocrine therapy shall be taken to include hormonal therapies such asselective estrogen reuptake inhibitors, selective estrogen receptordownregulators, aromatase inhibitors and ovarian ablation. Saidendocrine treatment may include administration of hormones or hormoneanalogs, synthetic hormones or other drugs to the patient, e.g.,tamoxifen, raloxifen and/or gosereline (tradename Zoladex®). In oneembodiment, the said endocrine treatment comprises the administration oftamoxifen or of tamoxifen and gosereline. Further, said endocrinetreatment may comprise the administration of an antiestrogen drugselected from the group comprising anastrozole, letrozole, exemestane,fulvestrant, toremifene and megasterol acetate. Said endocrine treatmentmay also comprise the administration of estrogen, progestin and/orgestagen.

The term “determining the expression level of a gene on a nonproteinbasis” relates to methods which are not focused on the secondary genetranslation products, i.e., proteins, but on other levels of the geneexpression, based on RNA and DNA analysis. In one embodiment of thisinvention the analysis uses mRNA including its precursor forms. Anexemplary determinable property is the amount of the estrogen receptoror progesterone receptor mRNA, i.e., of the ESR1, ESR2 and/or PGR gene.It may also include the detection of DNA amplification of the respectivegene.

The term “expression level” refers, e.g., to a determined level of geneexpression. The term “pattern of expression levels” refers to adetermined level of gene expression compared either to a reference gene,e.g., housekeeper, or inversely regulated genes, or to a computedaverage expression value, e.g., in DNA-chip analyses. A pattern is notlimited to the comparison of two genes but is more related to multiplecomparisons of genes to reference genes or samples. A certain “patternof expression levels” may also result and be determined by comparisonand measurement of several genes disclosed hereafter and display therelative abundance of these transcripts to each other. Expression levelsmay also be assessed relative to expression in different tissues, e.g.,expression of a gene in cancerous tissue vs. noncancerous tissue.

Alternatively, a differentially expressed gene disclosed herein may beused in methods for identifying reagents and compounds and uses of thesereagents and compounds for the treatment of cancer as well as methods oftreatment. The differential regulation of the gene is not limited to aspecific cancer cell type or clone, but rather displays the interplay ofcancer cells, muscle cells, stromal cells, connective tissue cells,other epithelial cells, fat cells, endothelial cells of blood vessels aswell as cells of the immune system, e.g., lymphocytes, macrophages,killer cells.

The term “RNA expression level” refers to a determined level of theconverted DNA gene sequence information into transcribed RNA, theinitial unspliced RNA transcript or the mature mRNA. RNA expression canbe monitored by measuring the levels of either the entire RNA of thegene or subsequences.

The term “pattern of RNA expression” refers to a determined level of RNAexpression compared either to a reference RNA or to a computed averageexpression value. A pattern is not limited to the comparison of two RNAsbut is more related to multiple comparisons of RNAs to reference RNAs orsamples. A certain “pattern of expression levels” may also result and bedetermined by comparison and measurement of several RNAs and display therelative abundance of these transcripts to each other. A “referencepattern of expression levels”, within the meaning of the invention shallbe understood as being any pattern of expression levels that can be usedfor the comparison to another pattern of expression levels. In anembodiment of the invention, a reference pattern of expression levelsis, e.g., an average pattern of expression levels observed in a group ofhealthy or diseased individuals, serving as a reference group.

The term “comparing the one or more expression levels(s)” expressionlevels” refers to the comparison of the expression levels, e.g., byarithmetical means, such as but not limited to the ratio of theexpression levels of two or more genes.

The terms “sample”, “biological sample”, or “clinical sample”, as usedherein, refer to a sample obtained from a patient. The sample may be ofany biological tissue or fluid. Such samples include, but are notlimited to, sputum, blood, serum, plasma, blood cells (e.g., whitecells), tissue, core or fine needle biopsy samples, cell-containing bodyfluids, free floating nucleic acids, urine, peritoneal fluid, andpleural fluid, liquor cerebrospinalis, tear fluid, or cells there from.Biological samples may also include sections of tissues such as frozenor fixed sections taken for histological purposes or microdissectedcells or extracellular parts thereof. A biological sample to be analyzedis tissue material from a neoplastic lesion taken by aspiration orpunctuation, excision or by any other surgical method leading to biopsyor resected cellular material. Such a biological sample may comprisecells obtained from a patient. The cells may be found in a cell “smear”in solid tumor material, in a lavage fluid, or in a body fluid. Thesample may be a processed sample, e.g., a sample, which has been frozen,fixed, embedded or the like. A sample that is usefully employed in thecontext of the present invention is a formaline fixed paraffin embedded(FFPE) sample. Preparation of FFPE samples are standard medical practiceand these samples can be conserved for long periods of time.

By “array” is meant an arrangement of addressable locations or“addresses” on a device. The locations can be arranged in twodimensional arrays, three dimensional arrays, or other matrix formats.The number of locations can range from several to at least hundreds ofthousands. Most importantly, each location represents an independentreaction site. Arrays include but are not limited to nucleic acidarrays, protein arrays and antibody arrays. A “nucleic acid array”refers to an array containing nucleic acid probes, such asoligonucleotides, polynucleotides or larger portions of genes. Thenucleic acid on the array can be rendered single stranded. Arrayswherein the probes are oligonucleotides are referred to as“oligonucleotide arrays” or “oligonucleotide chips.” A “microarray,”herein also refers to a “biochip” or “biological chip”, an array ofregions having a density of discrete regions of at least about 100/cm,and can be usefully employed as well having at least about 1000/cm, aswell-understood by those skilled in the art. The regions in a microarrayhave typical dimensions, e.g., diameters, in the range of between about10-250 μm, and are separated from other regions in the array by aboutthe same distance.

The term “oligonucleotide” refers to a relatively short polynucleotide,including, without limitation, single-stranded deoxyribonucleotides,single- or double-stranded ribonucleotides, RNA:DNA hybrids anddouble-stranded DNAs. Oligonucleotides can be single-stranded DNA probeoligonucleotides. Moreover, in context of applicable detectionmethodologies, the term “oligonucleotide” also refers to nucleotideanalogues such as PNAs and morpholinos.

The terms “modulated” or “modulation” or “regulated” or “regulation” and“differentially regulated” as used herein refer to both upregulation,i.e., activation or stimulation, e.g., by agonizing or potentiating, anddown regulation, i.e., inhibition or suppression, e.g., by antagonizing,decreasing or inhibiting.

The terms “primer”, “amplification primer”, “probes” and “labeledprobes”, within the meaning of the invention, shall have the ordinarymeaning of each term as is well known to the person skilled in the artof molecular biology. In the context of many embodiments of the presentinvention, these terms shall be understood as being polynucleotidemolecules having a sequence identical, complementary, homologous, orhomologous to the complement of regions of a target polynucleotide whichis to be detected or quantified. In yet another embodiment, nucleotideanalogues are also comprised for usage as primers and/or probes. Probetechnologies used for kinetic or real time PCR applications include,e.g., PCR systems generally, such as TaqMan® systems obtainable at RocheMolecular Diagnostics, extension probes such as Scorpion® Primers, DualHybridisation Probes, Amplifluor® obtainable at Chemicon International,Inc, or Minor Groove Binders. Probes can be surface bound, either on achip or on beads, and then be used as a microarray.

The phrase “response”, “therapeutic success”, or “response to therapy”refers in the neoadjuvant, adjuvant and palliative chemotherapeuticsetting to the observation of a defined tumor free or recurrence free orprogression free survival time (e.g., two years, four years, five years,ten years). This time period of disease-free, recurrence-free orprogression-free survival may vary among the different tumor entitiesbut is sufficiently longer than the average time period in which most ofthe recurrences appear. In a neoadjuvant and palliative therapymodality, response may additionally be monitored by measurement of tumorshrinkage and regression due to apoptosis and necrosis of the tumor massor reduced blood supply due to altered angiogenic events.

The term “recurrence” or “recurrent disease” includes distant metastasisthat can appear even many years after the initial diagnosis and therapyof a tumor, or local events such as infiltration of tumor cells intoregional lymph nodes, or occurrence of tumor cells at the same site andorgan of origin within an appropriate time.

“Prediction of recurrence” or “prediction of therapeutic success” doesrefer to the methods described in this invention, wherein a tumorspecimen is analyzed for, e.g., its gene expression, genomic statusand/or histopathological parameters (such as TNM and Grade) and/orimaging data and furthermore classified based on correlation of theexpression pattern to known ones from reference samples. Thisclassification may either result in the statement that such given tumorwill develop recurrence and therefore is considered as a“non-responding” tumor to the given therapy, or may result in aclassification as a tumor with a prolonged disease free post therapytime.

The term “marker” or “biomarker” refers to a biological molecule, e.g.,a nucleic acid, peptide, protein, hormone, etc., whose presence orconcentration can be detected and correlated with a known condition,such as a disease state or a combination of these, e.g., by amathematical algorithm.

The term “marker gene” as used herein, refers to a differentiallyexpressed gene whose expression pattern may be utilized as part of apredictive, prognostic or diagnostic process in malignant neoplasia orcancer evaluation, or which, alternatively, may be used in methods foridentifying compounds useful for the treatment or prevention ofmalignant neoplasia and gynecological cancer in particular. A markergene may also have the characteristics of a target gene.

“Target gene”, as used herein, refers to a differentially expressed geneinvolved in cancer, e.g., lung cancer, in a manner in which modulationof the level of the target gene expression or of the target gene productactivity may act to ameliorate symptoms of malignant neoplasia. A targetgene may also have the characteristics of a marker gene.

The term “receptor”, as used herein, relates to a protein on the cellmembrane or within the cytoplasm or cell nucleus that binds to aspecific molecule (a ligand), such as a neurotransmitter, hormone, orother substance, especially a hormone as estrogen, and initiates thecellular response. Ligand-induced changes in the behavior of receptorproteins result in physiological changes that constitute the biologicalactions of the ligands.

The term “signaling pathway” is related to any intra- or intercellularprocess by which cells converts one kind of signal or stimulus intoanother, most often involving ordered sequences of biochemical reactionsout- and inside the cell, that are carried out by enzymes and linkedthrough hormones and growth factors (intercellular), as well as secondmessengers (intracellular), the latter resulting in what is thought ofas a “second messenger pathway”. In many signaling pathways, the numberof proteins and other molecules participating in these events increasesas the process emanates from the initial stimulus, resulting in a“signal cascade” and often results in a relatively small stimuluseliciting a large response. In particular, the term “signaling pathways”relates to processes located upstream or downstream of a hormonereceptor, e.g., a ligand binding said receptor, or an intracellularsignaling cascade activated by said receptor.

The term “small molecule”, as used herein, is meant to refer to acompound which has a molecular weight of less than about 5 kD; notuncommonly, the small molecule employed in the context of the presentinvention is less than about 4 kD. Small molecules can be nucleic acids,peptides, polypeptides, peptidomimetics, carbohydrates, lipids or otherorganic (carbon-containing) or inorganic molecules. Many pharmaceuticalcompanies have extensive libraries of chemical and/or biologicalmixtures, often fungal, bacterial, or algal extracts, which can bescreened with any of the assays of the invention to identify compoundsthat modulate a bioactivity.

When used in reference to a single-stranded nucleic acid sequence, theterm “substantially homologous” refers to any probe that can hybridize(i.e., it is the complement of) the single-stranded nucleic acidsequence under conditions of low stringency as described above.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids.

The term “hybridization based method”, as used herein, refers to methodsimparting a process of combining complementary, single-stranded nucleicacids or nucleotide analogues into a single double stranded molecule.Nucleotides or nucleotide analogues will bind to their complement undernormal conditions, so two perfectly complementary strands will bind toeach other readily. In bioanalytics, very often labeled, single strandedprobes are in order to find complementary target sequences. If suchsequences exist in the sample, the probes will hybridize to saidsequences which can then be detected due to the label. Otherhybridization based methods comprise microarray and/or biochip methods.Therein, probes are immobilized on a solid phase, which is then exposedto a sample. If complementary nucleic acids exist in the sample, thesewill hybridize to the probes and can thus be detected. These approachesare also known as “array based methods”. Yet another hybridization basedmethod is PCR, which is described below. When it comes to thedetermination of expression levels, hybridization based methods may forexample be used to determine the amount of mRNA for a given gene.

The term “a PCR based method” as used herein refers to methodscomprising a polymerase chain reaction (PCR). This is an approach forexponentially amplifying nucleic acids, like DNA or RNA, via enzymaticreplication, without using a living organism. As PCR is an in vitrotechnique, it can be performed without restrictions on the form of DNA,and it can be extensively modified to perform a wide array of geneticmanipulations. When it comes to the determination of expression levels,a PCR based method may for example be used to detect the presence of agiven mRNA by (1) reverse transcription of the complete mRNA pool (theso called transcriptome) into cDNA with help of a reverse transcriptaseenzyme, and (2) detecting the presence of a given cDNA with help ofrespective primers. This approach is commonly known as reversetranscriptase PCR (rtPCR). The term “PCR based method” comprises bothend-point PCR applications as well as kinetic/real time PCR techniquesapplying special fluorophors or intercalating dyes which emitfluorescent signals as a function of amplified target and allowmonitoring and quantification of the target. Quantification methodscould be either absolute by external standard curves or relative to acomparative internal standard.

The term “method based on the electrochemical detection of molecules”relates to methods which make use of an electrode system to whichmolecules, particularly biomolecules like proteins, nucleic acids,antigens, antibodies and the like, bind under creation of a detectablesignal. Such methods are for example disclosed in WO0242759, WO0241992and WO02097413 filed by the applicant of the present invention, thecontent of which is incorporated by reference herein. These detectorscomprise a substrate with a planar surface which is formed, for example,by the crystallo-graphic surface of a silicon chip, and electricaldetectors which may adopt, for example, the shape of inter digitalelectrodes or a two dimensional electrode array. These electrodes carryprobe molecules, e.g., nucleic acid probes, capable of bindingspecifically to target molecules, e.g., target nucleic acid molecules.The probe molecules are for example immobilized by a Thiol-Gold-binding.For this purpose, the probe is modified at its 5′- or 3′-end with athiol group which binds to the electrode comprising a gold surface.These target nucleic acid molecules may carry, for example, an enzymelabel, like horseradish peroxidase (HRP) or alkaline phosphatase. Afterthe target molecules have bound to the probes, a substrate is then added(e.g., α-naphthyl phosphate or 3.3′ 5.5′-tetramethylbenzidine which isconverted by said enzyme, particularly in a redox-reaction. The productof said reaction, or a current generated in said reaction due to anexchange of electrons, can then be detected with help of the electricaldetector in a site specific manner.

The term “nucleic acid molecule” is intended to indicate any single- ordouble stranded nucleic acid and/or analogous molecules comprising DNA,cDNA and/or genomic DNA, RNA, such as, for example, mRNA, peptidenucleic acid (PNA), locked nucleic acid (LNA) and/or Morpholino.

The term “stringent conditions” relates to conditions under which aprobe will hybridize to its target subsequence, but to no othersequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (Tm) forthe specific sequence at a defined ionic strength and pH. The Tm is thetemperature (under defined ionic strength, pH and nucleic acidconcentration) at which 50% of the probes complementary to the targetsequence hybridize to the target sequence at equilibrium. (As the targetsequences are generally present in excess, at Tm, 50% of the probes areoccupied at equilibrium). Typically, stringent conditions will be thosein which the salt concentration is less than about 1.0 M Na ion,typically about 0.01 to 1.0 M Na ion (or other salts) at pH 7.0 to 8.3and the temperature is at least about 30° C. for short probes (e.g., 10to 50 nucleotides) and at least about 60° C. for longer probes.Stringent conditions may also be achieved with the addition ofdestabilizing agents, such as formamide and the like.

The term “fragment of the nucleic acid molecule” is intended to indicatea nucleic acid comprising a subset of a nucleic acid molecule accordingto one of the claimed sequences. The same is applicable to the term“fraction of the nucleic acid molecule”.

The term “variant of the nucleic acid molecule” refers herein to anucleic acid molecule which is substantially similar in structure andbiological activity to a nucleic acid molecule according to one of theclaimed sequences.

The term “homologue of the nucleic acid molecule” refers to a nucleicacid molecule the sequence of which has one or more nucleotides added,deleted, substituted or otherwise chemically modified in comparison to anucleic acid molecule according to one of the claimed sequences,provided always that the homologue retains substantially the samebinding properties as the latter.

The term “derivative” as used herein, refers to a nucleic acid moleculethat has similar binding characteristics to a target nucleic acidsequence as a nucleic acid molecule according to one of the claimedsequences

The term “hybridizing counterparts” as used herein, refers to a nucleicacid molecule that is capable of hybridizing to a nucleic acid moleculesunder stringent conditions.

The term “anamnesis” relates to patient data gained by a physician orother healthcare professional by asking specific questions, either ofthe patient or of other people who know the person and can give suitableinformation (in this case, it is sometimes called heteroanamnesis), withthe aim of obtaining information useful in formulating a diagnosis andproviding medical care to the patient.

This kind of information is called the symptoms, in contrast withclinical signs, which are ascertained by direct examination.

The term “etiopathology” relates to the course of a disease, that is itsduration, its clinical symptoms, and its outcome.

As used herein, the term “repair mechanisms related therewith” refers tocellular repair enzymes the expression of which correlates with theexpression of at least one of said hormone receptors selected from thegroup comprising estrogen receptor, progesterone receptor and/orandrogen receptor. A low ratio of hormone receptor versus EMT marker isgenerally correlated to an unfavorable outcome. Further, an decreased ordownregulated expression of said hormone receptor indicates that thepatient has lower PARP1 expression levels and higher angiogenicactivities and therefore benefit from a specific mode of therapy, inparticular treatments comprising targeting repair mechanisms andangiogenic activities selected from the group comprising PARP1, VEGFRs,PDGFRs, and/or their ligands and/or their respective signaling pathways.

OBJECT OF THE INVENTION

It is one object of the present invention to provide biological markersallowing one skilled in the medical arts to predict outcome of cancerpatients by providing prognostic and/or predictive informationconcerning the therapeutic outcome of a given treatment includingsurgery, systemic and/or local application of chemotherapeutic and/orendocrine agents as well as antibody based, nucleic acid based and/orsmall molecule based strategies.

It is another object of the present invention to provide a method forpredicting a clinical response of cancer to a given treatment based ontissue analysis before, during or after therapy.

These objects are met with the methods and means according to theindependent claims of the present invention.

SUMMARY OF THE INVENTION

Before the invention is described in detail, it is to be understood thatthis invention is not limited to the particular component parts of theprocess steps of the methods described as such methods may vary. It isalso to be understood that the terminology used herein is for purposesof describing particular embodiments only, and is not intended to belimiting. It must be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include singularand/or plural referents unless the context clearly dictates otherwise.It is also to be understood that plural forms include singular and/orplural referents unless the context clearly dictates otherwise. It ismoreover to be understood that, in case parameter ranges are given whichare delimited by numeric values, the ranges are deemed to include theselimitation values.

In its most general term, the invention relates to a method ofclassifying a sample of a patient who suffers from or is at risk ofdeveloping cancer, said method comprising the steps of determining insaid sample from said patient, on a non-protein basis, the expressionlevel of at least one gene in said sample encoding for a hormonereceptor selected from the group consisting of an estrogen receptor, aprogesterone receptor, and an androgen receptor, comparing the one ormore expression level(s) determined with one or more expression level(s)of one or more reference genes and thereby forming a pattern ofexpression level(s); and classifying the sample of said patient from theoutcome of the comparison into one of at least two classifications.

The method thus allows predicting a clinical response towards a givenmode of treatment. An increased or upregulated expression of saidhormone receptor is generally correlated to a favorable outcome.Further, an increased or upregulated expression of said hormone receptorindicates that the patient can benefit from a specific mode of therapy,in particular a treatment targeting at least one hormone receptorselected from the group comprising estrogen receptor, progesteronereceptor and/or androgen receptor, or targeting their respectivesignaling pathways, and/or a treatment targeting repair mechanismsrelated therewith.

As used herein, the term “signaling pathways” relates to processeslocated upstream or downstream of the said receptor, e.g., a ligandbinding said receptor, or an intracellular signaling cascade activatedby said receptor.

In a more specific term, the invention relates to a method of predictingan outcome of disease in a patient suffering from cancer wherein theexpression level of at least one gene encoding for a hormone receptorselected from the group consisting of an estrogen receptor, aprogesterone receptor, and an androgen receptor is determined on anonprotein basis and one gene selected from the group ofEpithelial-Mesenchymal-Transition factors comprising SNAI1, SNAI2 and/orSNAI3. The expression levels are set into a ratio thereby abrogating theneed of housekeeping or reference genes. This enables single welldetection of all relevant genes by multiplexing and eliminates problemsarising from variations (pipetting, enzyme reaction, fluorescencescanning, etc.). Moreover, it lowers costs and increases throughput ofthe diagnostic workflow with finite resources (sample amount, reagentcosts, capacity utilization). A high ratio of hormone receptor versusEMT marker is generally correlated to a favorable outcome. Further, anincreased or upregulated expression of said hormone receptor indicatesthat the patient can benefit from a specific mode of therapy, inparticular treatment comprising targeting hormone receptors selectedfrom the group comprising estrogen receptor, progesterone receptorand/or androgen receptor or targeting the respective hormones and/ortheir respective signaling pathways.

As used herein, the term “repair mechanisms related therewith” refers tocellular repair enzymes the expression of which correlates with theexpression of at least one of said hormone receptors selected from thegroup comprising estrogen receptor, progesterone receptor and/orandrogen receptor. A low ratio of hormone receptor versus EMT marker isgenerally correlated to an unfavorable outcome. Further, an decreased ordownregulated expression of said hormone receptor indicates that thepatient has lower PARP1 expression levels and higher angiogenicactivities and therefore benefit from a specific mode of therapy, inparticular treatments comprising targeting repair mechanisms andangiogenic activities selected from the group comprising PARP1, VEGFRs,PDGFRs, and/or their ligands and/or their respective signaling pathways.

By way of illustration and not by way of limitation said signalingactivities comprise receptor tyrosine kinase signaling, e.g., viaepidermal growth factor receptor (EGFR) family members, vascularendothelial growth factor receptor (VEGFR) signaling, Fibroblast GrowthFactor Receptor (FGFR) family members, Platelet Derived Growth FactorReceptor (PDGFR) family members, c-KIT, a proto-oncogene encoding areceptor tyrosine kinase, or Mesenchymal epithelial transition factor(c-Met); WNT signaling; Notch signaling; Hedgehog signaling;Transforming growth factor-beta (TGF-beta)/SMAD signaling and nuclearfactor-kappa B (NFkB) signaling.

In particular, the invention relates to the method and kit specified inthe claims. As specific embodiments of the invention, herein disclosedare the invention according to the following embodiments:

A first aspect of the invention is directed to a method of classifying asample of a patient suffering from or at risk of developing a lungcancer, said method comprising the steps of:

-   -   a. determining in said sample from said patient, on a non        protein basis, the expression level of at least one gene        encoding for a hormone receptor selected from the group        comprising estrogen receptor, progesterone receptor and/or        androgen receptor in said sample;    -   b. comparing the pattern of expression level(s) determined in        step (a) with one or several reference pattern(s) of expression        levels; and    -   c. classifying the sample of said patient from the outcome of        the comparison in step (b) into one of at least two        classifications.

A second aspect of the invention is directed to a method for predictinga clinical response of a patient suffering from or at risk of developinga lung cancer towards a given mode of treatment, said method comprisingthe steps of:

-   -   a. determining in a sample from said patient, on a non protein        basis, the expression level of at least one gene encoding for a        hormone receptor selected from the group comprising estrogen        receptor, progesterone receptor and/or androgen receptor in said        sample;    -   b. comparing the pattern of expression level(s) determined in        step (a) with one or several reference pattern(s) of expression        levels; and    -   c. predicting therapeutic success for said given mode of        treatment in said patient from the outcome of the comparison in        step (b).

A third aspect the invention is directed to a method of predicting aclinical response towards a given mode of cancer treatment orclassifying a sample of a patient who suffers from or being at risk ofdeveloping cancer, said method comprising the steps of:

-   -   a. determining in said sample from said patient, on a non        protein basis, the expression level of at least one gene        encoding for a hormone receptor selected from the group        comprising estrogen receptor, progesterone receptor and/or        androgen receptor in said sample;    -   b. comparing the one or more expression level(s) determined in        step (a) with one or more expression level(s) of one or more        reference genes and thereby forming a pattern of expression        level(s); and    -   c. classifying the sample of said patient from the outcome of        the comparison in step (b) into one of at least two        classifications.

In one embodiment, the mode of treatment based on the classification instep (c) comprises an endocrine treatment by targeting hormone receptorsselected from the group comprising estrogen receptor, progesteronereceptor and/or androgen receptor or their respective signaling pathwaysand/or a treatment targeting repair mechanisms related therewith.

In another embodiment, said endocrine treatment is a hormonal treatmentand/or antihormonal treatment. In yet another embodiment, said endocrinetreatment comprises the administration of tamoxifen. In anotherembodiment, said endocrine treatment is intended to be given as hormonereplacement therapy (HRT) in peri- or postmenopausal women. In yetanother embodiment, the gene encoding for the estrogen receptor is ESR1.

In another embodiment, the upregulated expression of said at least onegene encoding for a hormone receptor selected from the group comprisingestrogen receptor, progesterone receptor and/or androgen receptordetermined in step (a) is indicative of a promising prediction asregards therapeutic success for said given mode of treatment.

In yet another embodiment, it is an intermediate upregulated expressionof said at least one gene encoding for a hormone receptor selected fromthe group comprising estrogen receptor, progesterone receptor and/orandrogen receptor determined in step (b) which is indicative of apromising prediction as regards therapeutic success for a therapeuticregimen targeting hormone receptors selected from the group comprisingestrogen receptor, progesterone receptor and/or androgen receptor. In anembodiment, said one or more reference gene(s) is at least onehousekeeping gene and/or at least one EMT marker gene.

In another embodiment, the at least one housekeeping gene is selectedfrom the group comprising RPL37A, GAPDH, RPL13 and/or HPRT1; and the atleast one EMT marker gene is selected from the group comprising SNAI1,SNAI2 and/or SNAI3.

In another embodiment, the comparison in step (b) of the method is a twogene ratio between the expression level of a hormone receptor and an EMTmarker gene, such as, for example, a ratio of ESR1 to SNAI2.

In another embodiment, said given mode of treatment acts on recruitmentof lymphatic vessels, angiogenesis, cell proliferation, cell survivaland/or cell motility, and/or comprises administration of achemotherapeutic agent.

In a further embodiment said given mode of treatment is selected fromthe group comprising chemotherapy, administration of small moleculeinhibitors, antibody based regimen, anti-proliferation regimen,pro-apoptotic regimen, pro-differentiation regimen, radiation and/orsurgical therapy.

The invention is also directed to a method of selecting a therapymodality for a patient afflicted with a lung cancer, said methodcomprising the steps of:

-   -   a. predicting from a biological sample from said patient, by the        method according to any one of the aforementioned numbered        paragraphs, therapeutic success for a plurality of individual        modes of treatment; and    -   b. selecting a mode of treatment which is predicted to be        successful in step (a).

And the invention is further directed to a method for adaptingtherapeutic regimen based on individualized risk assessment for apatient suffering from or at risk of developing a lung cancer,comprising the steps of:

-   -   a. determining in a biological sample from said patient, on a        non protein basis, the expression level of at least one gene        encoding for a hormone receptor selected from the group        comprising estrogen receptor, progesterone receptor and/or        androgen receptor in said sample;    -   b. comparing the pattern of expression level(s) determined in        step (a) with one or several reference pattern(s) of expression        levels; and    -   c. implementing therapeutic regimen targeting hormone receptors        selected from the group comprising estrogen receptor,        progesterone receptor and/or androgen receptor or signaling        pathways in said patient from the outcome of the comparison in        step (b).

In a number of the recited embodiments, said expression level(s) isdetermined by

-   -   a. a hybridization based method;    -   b. a PCR based method;    -   c. a method based on the electrochemical detection of particular        molecules, and/or    -   d. an array based method.

In another embodiment said expression level is determined by reversetranscriptase polymerase chain reaction of RNA transcripts.

In yet another embodiment said expression level is determined informalin and/or paraffin fixed tissue samples of the RNA transcripts.

In a further embodiment, the sample is treated with silica-coatedmagnetic particles and a chaotropic salt, for purification of thenucleic acids contained in said sample prior to the determination instep (a).

In another embodiment, the upregulated expression level of said at leastone gene encoding for a hormone receptor selected from the groupcomprising estrogen receptor, progesterone receptor and/or androgenreceptor is related to a favorable outcome, in particular to prolongedsurvival.

In yet another embodiment, said cancer displays characteristics of or isan adenocarcinoma.

It yet another embodiment, it could also be a non-carcinogen neoplasticdisease. In another embodiment, the cancer is selected from the groupconsisting of a lung cancer, a non-small cell lung cancer (NSCLC), anovarian cancer, a breast cancer, and a prostate cancer.

In another embodiment, the pattern of expression level(s) determined instep (a) is correlated with said patient's data, said data beingselected from the group consisting of etiopathology data, clinicalsymptoms, anamnesis data and/or data concerning the therapeutic regimen.

The invention is further directed to a kit useful for carrying out amethod of any one of the aforementioned numbered paragraphs, comprisingat least a pair of gene specific primers and/or probes each having asequence sufficiently complementary to at least one gene or genefragments or genomic nucleic acid sequence encoding for a at least onegene coding for a hormone receptor selected from the group comprisingestrogen receptor, progesterone receptor and/or androgen receptor forquantifying the expression of said at least one gene or gene fragment orgenomic nucleic acid sequence, and/or their fractions, variants,homologues, derivatives, fragments, complements, hybridizingcounterparts, or molecules sharing a sequence identity of at least about70%, of at least about 75%, of at least about 80%, of at least about85%, of at least about 90%, of at least about 95%, of at least about97%.

It is again pointed out that all details of the methods and kitsdescribed are not limited to their application in lung cancer but alsoto other types of cancer. Thus, lung cancer is only the example ofchoice. Up to now the prognostic and predictive role of hormonereceptors in lung cancer selected from the group comprising estrogenreceptor (ESR), progesterone receptor (PGR) and/or androgen receptor(AR) in lung cancer has not been shown. Accordingly no endocrinetreatment options are offered to lung cancer patients in the neoadjuvantor adjuvant setting.

The hormone receptor RNA expression of ESR1, ESR2, PGR, AR in fresh andfixed tissue biopsy samples and tumor resectates of stage III and IVsmall cell and non-small cell lung cancer patients from anon-stratified, population based cohort treated with chemotherapy wereanalyzed.

Surprisingly, it was found that the expression level of a gene encodingfor a hormone receptor selected from the group comprising estrogenreceptor, progesterone receptor and/or androgen receptor has prognosticand/or predictive value in lung cancer.

In this regard it is to be understood, that the analysis of estrogenreceptor and progesterone receptor status on protein basis has turnedout to be inferior to the detection of genes coding for estrogenreceptor on RNA basis, as the determination of estrogen receptor byimmune histochemistry fails to have prognostic value for lung cancer.This has been experimentally confirmed by the inventors in a very samecohort of patients, where the diagnostic value of estrogen and/orprogesterone receptor expression determination by kinetic PCR (kPCR)methods has been proven.

The validity of these findings have been shown by independentmeasurements of fresh tissue biopsies and resectates by array analysisand also by PCR based analysis of clinical routine material, i.e.,formalin fixed and paraffin embedded (=FFPE) tissues.

Moreover, it was established, for the first time, to use the expressionlevel of a gene encoding for hormone receptors selected from the groupcomprising estrogen receptor, progesterone receptor and/or androgenreceptor for the decision whether a given therapy is the most promisingtherapy for the respective patient having lung cancer or if treatmentmodalities should be altered. In particular, the method disclosed hereinis highly prognostic in the identical samples of a patient cohort wherethe state of the art technology, i.e., immunohistochemistry (=IHC),clearly fails to have any prognostic information.

The prediction of therapeutic success or the investigation of theresponse to a treatment can be performed at time of first biopsy orafter surgery, at a stage in which other methods cannot provide therequired information on the patient's response to chemotherapy. Hencethe current invention also provides means to decide even shortly aftertumor surgery whether or not a certain mode of chemo-therapy is likelyto be beneficial to the patient's health and/or whether to maintain orchange the applied mode of chemotherapy treatment. This is of particularimportance as the decision which systemic therapy to apply first is ofoutmost importance for survival, development of resistance and thereforealso for subsequent treatment strategies. Also the overall status ofpatients is usually best at initial diagnosis and therefore allows toapply more complex and/or aggressive treatment options at intended. Thisnot only holds true for chemotherapeutic strategies but is also ofimportance for generally less toxic strategies, such as anti-angiogenictreatments as exemplified by application of Bevacizumab (tradenameAvastin®), Sunitinib (tradename Sutent®) or Sorafenib (tradenameNexavar®). The reason for this is in part the extensive surgery beingnecessary for lung cancer, which inter alias increases the risk ofbleeding and intraoperative or postoperative death.

According to the superiority of non-protein based determination ofhormone receptor status selected from the group comprising estrogenreceptor, progesterone receptor and/or androgen receptor status, themethod should substitute currently available measurements, or used inaddition to currently available tests or histopathological parameter tomake diagnosis more accurate.

Furthermore, the method according to the invention may be applied inneoadjuvant, adjuvant and metastatic settings. Importantly, theinventors have found that hormone receptors such as estrogen,progesterone and/or androgen receptor are useful for prediction based onuntreated tumor samples but also prognostic for treated tumor samples.

The inventor suggests, for the first time, to use the expression levelof at least one gene encoding for estrogen receptor for the decisionwhether a therapeutic regimen targeting a hormone receptor, e.g.,estrogen receptor, in other words, an endocrine therapy, could bebeneficial in a lung cancer patient. This particularly not onlycomprises different kinds of hormone antagonists or enzyme inhibitorsblocking steps of the estrogen biosynthesis but also the usage ofhormone agonists, e.g., estrogen, as this could accelerate hormonalcontrol of deregulated cancer cell activities and/or sensitize towardsother therapeutic options such as chemotherapy.

It is yet another embodiment of the invention to combine the informationof the mRNA expression level of at least one gene encoding for hormonereceptors such as estrogen, progesterone and/or androgen receptor withthe level of microRNAs regulating the stability and/or translation ofrespective mRNAs. By way of illustration and not by way of limitationthis may mean that tumors expressing intermediate levels of ESR1 mRNA doworse, when higher levels of microRNAs downregulate ESR1 proteinexpression, compared to tumors expressing intermediate levels of ESR1mRNA that do not display microRNA expression of respective microRNAs.

It is yet another embodiment of the invention to combine the informationof the mRNA expression level of at least one gene encoding for hormonereceptors such as estrogen, progesterone and/or androgen receptor withthe expression level of CYP19A1, which is responsible for the conversionof androgens to estrogens is expressed not only in gonads and adrenalsbut also in many other tissues, including normal lungs and lung cancersand therefore produces hormone receptor ligands in proximity to theneoplastic tissue. This contributes to the finding being part of thisinvention, that the determination of hormone receptors is not onlyuseful in women but also in men and therefore of diagnostic andtherapeutic importance in both genders.

It is yet another embodiment of the present invention to provide amethod for predicting the development of resistance to therapeuticintervention of a patient suffering from lung cancer to a giventreatment.

In this context it is of note that it is part of this invention to usethe described method to stratify patients, which may benefit fromhormonal treatments.

It is yet another embodiment that it is part of this invention to usethe described method to stratify patients, which may benefit from PARPInhibition.

Another embodiment of the present invention provides a method tostratify patients for systemic treatments other than chemotherapy in aneoadjuvant, adjuvant or palliative setting. In one embodiment, thesealternative treatment options comprise antibody based or small moleculebased treatment. Notably, treatment in the context of the presentinvention includes endocrine treatment options.

Moreover, the method according to the invention may help to detect thosetumors which are probably more susceptible to endocrine treatment thanto a chemotherapeutic regimen. These tumors have so far remainedundetected with methods from the state of the art. Particularly thedetermination of ESR1 status by IHC is not part of the current standardof care as it does not provide any prognostic information. Thereby theendocrine options have been neglected for treatment of lung cancer andovarian cancer.

The present inventive method includes the step of assessing theexpression level of at least one gene encoding for an estrogen receptorthat is selected from the group consisting of ESR1, ESR2, progesteronereceptor, PGR, and androgen receptor. This assessment is usefullyemployed in the context of the present invention for deciding whether atherapeutic regimen targeting signaling pathways, as specified above orotherwise, could be beneficial in that patient.

The inventor suggests moreover to use the expression level of at leastone gene encoding for an estrogen receptor, such as, for example, onethat is selected from the group consisting of ESR1, ESR2, a progesteronereceptor, PGR, and an androgen receptor for the decision whether atherapeutic regimen targeting matrix metalloproteinases could bebeneficial in that patient. These regimens comprise therapeuticsblocking the protease activity of MMP1, MMP2, MMP7, MMP9 and/or MMPlO.

The inventor suggests moreover to use the expression level of at leastone gene encoding for an estrogen receptor, such as one selected fromthe group consisting of ESR1, ESR2, a progesterone receptor, PGR, and anandrogen receptor for the decision whether a therapeutic regimentargeting repair mechanism could be beneficial in that patient. Theseregimen comprise therapeutics blocking the PARP1 gene product.

The inventor suggests moreover to use the expression level of at leastone gene encoding for an estrogen receptor, such as, for example, andwithout limitation intended, one selected from the group consisting ofESR1, ESR2, a progesterone receptor, PGR, and an androgen receptor forthe decision whether a therapeutic regimen affecting bone metabolism,such as bisphosphonates, and/or antibody regimen having similarproperties by attacking the RANKL system, such a denosumab, could bebeneficial in that patient.

The inventor has found that the balance between hormone receptors andstem cell activities or SNAI factors is indicative of tumor outcome inlung cancer. However the inventors suggests that this balance is notlimited to lung cancer but rather a general tumor principle. The presentinvention moreover sets forth a method to use the expression level of atleast one gene encoding for an estrogen receptor, such as, for example,and without limitation intended, one selected from the group consistingof ESR1, ESR2, a progesterone receptor, PGR, and an androgen receptorfor the decision whether targeted therapy such as an anti-tyrosinekinase regimen may be effective. This relates to the finding, that theabsence or low activity of hormone receptors relates to more aggressivetumors characterized by, e.g., elevated EGFR family and VEGFR familyactivities. The inventor suggests moreover to use the expression levelof at least one gene encoding for an estrogen receptor, such as oneselected from the group consisting of ESR1, ESR2, a progesteronereceptor, PGR, and an androgen receptor for the decision whether atyrosine kinase inhibitor could be beneficial in a patient suffering anadenocarcinoma bearing mutated tyrosine kinase expression. The mutatedtyrosine kinase in the patient that may be benefited by the presentinvention is EGFR of c-Met.

In one embodiment of the invention, said given mode of chemotherapy istargeted therapy such as small molecule inhibitors like Sunitinib(tradename Sutent®), Sorafenib (tradename Nexavar®), Lapatinib(tradename Tykerb®) and/or therapeutic antibodies, e.g., Bevacizumab(tradename Avastin®) or cetuximab (tradename Erbitux®).

However, other treatments related to signaling pathways which fall underthe scope of the present invention comprise the administration of BAY43-9005, target receptors are VEGFR-2, VEGFR-3, c-KIT, PDGFR-B, RET andRaf-Kinase), BAY 57-9352 (target receptor is VEGFR-2), Sunitinib(tradename Sutent®, target receptors are VEGFR-I, VEGFR-2 and PDGFR),AG13925 (target receptors are VEGFR-I and VEGFR-2), AGO 13736 (targetreceptors are VEGFR-I and VEGFR-2), AZD2171 (target receptors areVEGFR-I and VEGFR-2), ZD6474 (target receptors are VEGFR-I, VEGFR-2 andVEGFR-3), PTK-787/ZK-222584 (target receptors are VEGFR-I and VEGFR-2),CEP-7055 (target receptors are VEGFR-I, VEGFR-2 and VEGFR-3), CP-547(target receptors are VEGFR-I and VEGFR-2), CP-632 (target receptors areVEGFR-I and VEGFR-2), GW786024 (target receptors are VEGFR-I, VEGFR-2and VEGFR-3), AMG706 (target receptors are VEGFR-I, VEGFR-2 andVEGFR-3), Imatinib mesylate (tradename Glivec®/Gleevec®, targetreceptors are bcr-abl and c-KIT), BMS-214662 (target enzyme is Rasfarnesyl transferase), CCI-779 (target enzyme is mTOR), RADOOO1(tradename Everolismus®, target enzyme is mTOR), CI-1040 (target enzymeis MEK), SU6668 (target receptors are VEGFR-2, PDGFR-B and FGFR-I),AZD6126, CP547632 (target receptors are VEGFRs), CP868596 GW786034(target receptors are PDGFRs), ABT-869 (target receptors are VEGFRs andPDGFRs), AEE788 (target receptors are VEGFRs and PDGFRs), AZD0530(target enzymes are src and abl), and CEP7055.

In another embodiment, the genes encoding for estrogen receptor areselected from the group consisting of ESR1 and ESR2. In one embodiment,the gene encoding for the estrogen receptor is ESR1.

Surprisingly, the inventors have found that the expression level of ESR1has good prognostic and/or diagnostic value in lung cancer when testedbefore treatment, which resembles the de novo hormone activity of thetumor tissue. More surprisingly, the inventors have found that thebenefit from chemotherapy was particularly striking in high grade and/orhigher size tumors expressing estrogen and progesterone receptors, whilethe response of estrogen or progesterone receptor negative tumorsremained to be poor. The prognostic value of hormone receptor activitywas particularly prominent in NSCLC and in women suffering lung cancer,thereby contributing to the known better prognosis of the tumors inthese lung cancer subgroups.

Therefore, the inventor suggests for the first time, to use theexpression level of ESR1 and/or ESR2 for the decision whether a giventherapy is the most promising therapy for lung cancer, or if treatmentmodalities should be altered. As the inventor does show by comparingwith the current standard techniques, these decisions cannot be drawnwith, e.g., IHC, as these techniques fail to determine the prognosticvalue of hormone receptors.

In another embodiment, the gene encoding for the progesterone receptoris PGR. In yet another embodiment, PGR is used for to decide ontreatment modalities.

Moreover surprisingly, the inventor has found that the expression levelof PGR has good prognostic and/or diagnostic value in lung cancer.

The inventor suggests for the first time, to use the expression level ofPGR for the decision whether a given therapy is the most promisingtherapy for lung cancer or if treatment modalities should be altered.

In another embodiment, the gene encoding for the androgene receptor isAR. In yet another embodiment, AR is used for to decide on treatmentmodalities.

Moreover surprisingly, the inventor has found that the expression levelof AR has prognostic and/or diagnostic value in lung cancer.

The inventors suggest for the first time, to use the expression level ofAR for the decision whether a given therapy is the most promisingtherapy for lung cancer or if treatment modalities should be altered.

In another embodiment the gene encoding for the aromatase is CYP19. Inyet another embodiment CYP19 is used for to decide on treatmentmodalities.

Moreover surprisingly, the inventors have found that the expressionlevel of CYP19 has prognostic and/or diagnostic value in lung cancer,particularly when combined with the expression level of hormonereceptors and, as one example, ESR1. The inventors suggest for the firsttime, to use the expression level of CYP19 and/or ESR1 for the decisionwhether a given therapy is the most promising therapy for lung cancer orif treatment modalities should be altered.

In another embodiment, the microRNA affecting the ESR1 expression is206, 221 and/or 222. In yet another embodiment, microRNA is used for todecide on treatment modalities.

Moreover surprisingly, the inventor has found that the microRNA hasprognostic and/or diagnostic value in lung cancer, particularly whencombined with the expression level of hormone receptors and, as oneuseful example, ESR1.

The inventors suggest for the first time, to use the expression level ofmicroRNA and/or ESR1 for the decision whether a given therapy is themost promising therapy for lung cancer or if treatment modalities shouldbe altered.

Importantly, the decision when to use altered treatment modalities suchas endocrine options can be influenced. These treatment modalities maybe applied before, during or after chemotherapy and/or surgery.

In another embodiment of the present invention, the methods of thepresent invention comprise comparing the level of mRNA expression ofESR1 and/or ESR2 and/or PGR and/or AR in a patient sample, and theaverage level of expression of ESR1 and/or ESR2 and/or PGR and/or AR ina sample from a control subject, e.g., a human subject without cancer.Comparison of the pattern of expression levels of ESR1 and/or ESR2and/or PGR and/or AR can also be performed on any other reference.

In another embodiment of the present invention, the methods of thepresent invention also comprise comparing the pattern of expressionlevels of mRNA of ESR1 and/or ESR2 and/or PGR and/or AR in anunclassified patient sample, and the pattern of expression levels ofESR1 and/or ESR2 and/or PGR and/or AR in a sample cohort comprisingpatients responding in different intensity to an administeredneoadjuvant, adjuvant and/or palliative cancer therapy.

In another embodiment of this invention, the expression of ESR1 and/orESR2 and/or PGR and/or AR can be utilized for discrimination ofresponders and non-responders to a given treatment, especially achemotherapeutic and/or endocrine intervention.

In another embodiment of the present invention, it is provided thatupregulated expression of said at least one gene encoding for a hormonereceptor selected from the group comprising estrogen receptor,progesterone receptor and/or androgen receptor, especially of the RNAtranscripts of ESR1, determined in step (b) is indicative of a promisingprediction as regards therapeutic success for a given mode of treatment.

Moreover, the combined analysis of estrogen, progesterone and androgenreceptors improved the diagnostic value of the single marker evaluation,i.e., just based on estrogen, progesterone or androgen receptor.

By correlation analysis, the inventors have found that overexpression ofESR1 in untreated tumor samples that are, for example, assessed by PCRanalysis, is an indicator for a good prognosis of lung cancer patientstreated by standard chemotherapy as indicated by prolonged disease freeand overall survival. Especially a high expression of ESR1 was found toprovide a good overall survival prognosis upon standard adjuvantchemotherapy. Also in the palliative chemotherapeutic setting theelevated expression level of estrogen receptors and progesteronereceptors was associated with increased response to endocrinetreatments. This indicates the direct link between treatment anddirectly related response, i.e., tumor shrinkage, whose assumption isdifficult to draw in the adjuvant setting. In other embodiments,intermediate expression of ESR1, for example assessed by PCR analysis,indicates poor prognosis of lung cancer patients treated by standardchemotherapy.

In another embodiment of the present invention, it is provided thathighly or intermediately upregulated expression of said at least onegene encoding for a hormone receptor selected from the group comprisingestrogen receptor, progesterone receptor and/or androgen receptordetermined in step (b) is indicative of a promising prediction asregards therapeutic success for a therapeutic regimen targeting hormonereceptors selected from the group comprising estrogen receptor,progesterone receptor and/or androgen receptor especially endocrinetreatment.

In another embodiment of the present invention, it is provided thathighly or intermediately upregulated expression of said at least onegene encoding for the estrogen receptor especially ESR1 determined instep (b) is indicative of a promising prediction as regards therapeuticsuccess for a therapeutic regimen targeting the estrogen receptor,especially endocrine treatment.

In yet another embodiment of the present invention, it is provided thathighly or intermediately upregulated expression of said at least onegene encoding for the estrogen receptor especially ESR1 determined instep (b) is indicative of increased risk of bone metastasis a promisingprediction as regards therapeutic success for a therapeutic regimentargeting the bone metabolism (such as bisphosphonates, denosumab).

In another embodiment of the present invention, it is provided thathighly or intermediately upregulated expression of said at least onegene encoding for the estrogen receptor especially ESR1 anddownregulated expression of said at least one gene encoding EMT markersespecially SNAIL2 simultaneous determined in step (b) is indicative of apromising prediction as regards therapeutic success for a therapeuticregimen targeting the estrogen receptor, especially endocrine treatment.

In another embodiment of the present invention, it is provided thatdownregulated expression of said at least one gene encoding EMT markersespecially SNAIL2 determined in step (b) is indicative of a promisingprediction as regards therapeutic success for a therapeutic regimentargeting the estrogen receptor, especially endocrine treatment.

For example, in the case of highly upregulated expression of said atleast one gene encoding for the estrogen receptor especially ESR1determined in step (b) the nodal status may provide additionalinformation with regard to outcome. In particular, if node negative theoutcome of patients with high expression of, e.g., ESR1 may be very good(i.e., above 95% survival), whereas if node positive, the outcome may beinferior (i.e., at about 80% survival), while still being clearlysuperior to bad prognosis at low expression of, e.g., ESR1 (i.e., at 22%survival). This means that patients with tumors exhibiting highexpression of, e.g., ESR1 still may have a benefit from additionalendocrine treatment.

In yet another embodiment of the present invention, it is provided thatlow expression of said at least one gene encoding for a hormone receptorselected from the group comprising estrogen receptor, progesteronereceptor and/or androgen receptor especially ESR1 indicates poorprognosis of lung cancer patients treated by standard chemotherapy.

Moreover this finding also enables to decide which patients shouldreceive other treatment options targeting signaling pathways, e.g.,small molecules.

In another embodiment of the present invention, it is provided that thepattern of expression level(s) determined in step (b) refers to a levelof gene expression compared to a reference selected from the groupcomprising RPL37A, GAPDH, CALM2, OAZ1 RPL13, and/or HPRT1. In anotherembodiment these reference genes are RPL37A, GAPDH and HPRT1. In yetanother embodiment, the reference genes are RPL37A and HPRT1. In anotherembodiment of the present invention, it is provided that said referenceor housekeeping gene is RPL37A.

Normalization to a housekeeping gene selected from the group comprisingRPL37A, GAPDH, RPL13, and/or HPRT1 can provide the advantage of a highlyreliable comparison. In another embodiment of the present invention, itis provided that the pattern of expression level(s) determined in step(b) refers to a level of gene expression compared to an anticorrelatedgene reference selected from the group comprising SNAIL1, SNAIL2, CDH11,MMP2. In an embodiment, these reference genes are SNAIL1.

In yet another embodiment of the present invention, it is provided thatsaid given mode of treatment acts on recruitment of lymphatic vessels,angiogenesis, cell proliferation, cell survival and/or cell motility,and/or comprises administration of a chemotherapeutic agent.

Furthermore, it is provided in an another embodiment of the presentinvention that said given mode of treatment is selected from the groupcomprising chemotherapy, administration of small molecule inhibitors,antibody based regimen, anti-proliferation regimen, pro-apoptoticregimen, pro-differentiation regimen, radiation and/or surgical therapy.In yet other embodiments said given mode of treatment may includeadministration of cis-Platin (tradename Cisplatin®).

Said chemotherapy may comprise the administration of at least one agentselected from the group comprising Cyclophosphamid (Endoxan®,Cyclostin®). Adriamycin (Doxorubicin) (Adriblastin®), BCNU (Carmustin)(Carmubris®), Busulfan (Myleran®), Bleomycin (Bleomycin®), Carboplatin(Carboplat®), Chlorambucil (Leukeran®), Cis-Platin (Cisplatin®),Platinex (Platib-Lastin®), Dacarbazin (DTIC®; Detimedac®), Docetaxel(Taxotere®), Epirubicin (Farmorubicin®), Etoposid (Vepesid®),5-Fluorouracil (Fluroblastin®, Fluorouracil®), Gemcitabin (Gemzar®),Ifosfamid (Holoxan®), Interferon alpha (Roferon®), Irinotecan (CPT 11,Campto®), Melphalan (Alkeran®), Methotrexat (Methotrexat®,Farmitrexat®), Mitomycin C (Mitomycin®), Mitoxantron (Novantron®),Oxaliplatin (Eloxatine®), Paclitaxel (Taxol®), Prednimustin (Sterecyt®),Procarbazin (Natulan®), Pemetrexed (Alimta®), Ralitrexed (Tomudex®),Topotecan (Hycantin®), Trofosfamid (Ixoten®), Vinblastin (Velbe®),Vincristin (Vincristin®), Vindesin (Eldisine®) and/or Vinorelbin(Navelbine®).

In other embodiments said given mode of treatment may be endocrinetreatment.

In a further aspect, the present invention provides a method ofselecting a therapy modality for a patient afflicted with lung cancer,said method comprising the steps of:

-   -   a. obtaining a biological sample from said patient;    -   b. predicting from said sample, by the described before,        therapeutic success for a plurality of individual modes of        treatment; and    -   c. selecting a mode of treatment which is predicted to be        successful in step (b).

It is of note, that the inventors have proven the validity of thedisclosed method in fresh tissue as well as fixed tissues. Also theinventors have shown the validity of the disclosed method in biopsies aswell as tumor resectates.

On the basis of the findings of the present invention a therapy can beselected, which is most promising for the individual patient.

In a further aspect, the present invention provides a method ofselecting a modality for a patient afflicted with lung cancer, saidmethod comprising the steps of:

-   -   a. obtaining a biological sample from said patient;    -   b. predicting from said sample, by the method described before,        diagnostic success for a plurality of individual modes of        imaging; and    -   c. selecting a mode of imaging which is predicted to be        successful in step (b).

On the basis of the findings of the present invention an imagingmodality can be selected, which is most promising for the individualpatient.

Here the inventor has shown for the first time that high or intermediateexpression of ESR1 is predictive for increased risk of bone metastasisin lung cancer patients.

Based on the ESR1, PGR, AR and or snail mRNA determination in theprimary tumor the subsequent imaging modality can be chosen for moreprecise staging and tailored treatment choice. Higher ESR1 expressionindicates a bone scan or application of labeled estrogen receptorligands (e.g., fluoridinated estradiol “[18F]FES”). Higher PGRexpression indicates a bone scan or application of labeled progesteronreceptor ligands (e.g., fluoridinated progrestin. Higher AR expressionindicates a bone scan or application of labeled androgen receptorligands (e.g., fluoridinated testosterone). Higher snail expressionindicates application of labeled Matrix-Metallo-Proteinase (=MMP)ligands (e.g., labeled MMP inhibitors, particularly for MMP2).

Higher risk of bone involvement in disease progression may indicatealtered treatment, e.g., by including bisphosphonates or antibodiesagainst RANKL (such as denosumab “Prolia®”) to treat metastatic spreadand recruitment of bone marrow derived precursor cells early on. This isthought to prevent disease progression and potentially prolong life.

In addition the inventor suggests, for the first time, to use theexpression level of a gene encoding for the estrogen receptor and/orprogesterone receptor, especially ESR1, for the decision whether or notchemotherapeutic treatment should be kept as treatment or if endocrinetreatment or treatment options targeting signaling pathways should beincluded as a treatment options. In yet another addition the inventorsuggests, for the first time, to use the expression level of a geneencoding for the estrogen receptor and/or progesterone receptor,especially ESR1, for the decision whether or not bone preservingtreatments should be included as a therapeutic option in lung cancer.

In this regard, the accurate detection of the expression level of ESR1enables to identify a subpopulation of tumors that overexpress ESR1 inan intermediate or slightly higher fashion, yet having a comparativelylow overexpression of ESR1 that cannot be resolved byimmunohistochemical techniques. This subpopulation may be particularlysensitive to endocrine treatment.

The methods of the invention maybe used to evaluate a patient before,during and after therapy, for example to evaluate the reduction in tumorburden.

In the method of the present invention the determination of geneexpression or the determination of the pattern of expression level isnot limited to any specific method, or to the detection of mRNA.

In the method according to the invention, said expression leveldetermined in step (b) can be determined by

-   -   a. a hybridization based method;    -   b. a PCR based method;    -   c. a method based on the electrochemical detection of particular        molecules, and/or    -   d. an array based method.

The above mentioned methods have in common that they are focused on thedetection of nucleic acids, particularly on the detection of mRNA, DNA,peptide nucleic acid (PNA), locked nucleic acid (LNA) and/or Morpholino.

Moreover, these methods provide the option that high qualitydeterminations can be done as multiplex assays in one reaction based onthe high specificity of the reagent design and performance.

Another advantage is that the method requires only small amounts ofbiological sample.

In yet another embodiment of the present invention, it is provided thatsaid expression level of the RNA transcripts is determined by reversetranscriptase polymerase chain reaction (RT-PCR).

The method according to the invention has the advantage that it works onparaffin embedded tissues. In yet another embodiment of the presentinvention, it is provided that said expression level of the RNAtranscripts is determined in formalin and/or paraffin fixed tissuesamples.

For this purpose, at least one fixative may be used in an embodimentwhich is selected from the group consisting of Neutral BufferedFormaline, Unbuffered Formaline, Glutaraldehyde, Ethanol, Acetone,Methanol, Methacarn, Carnoy's fixative, AFA-Fixative (Formaldehyde,Ethanol and acetic acid), Pen-Fix (alcoholic formalin fixative),Glyo-Fixx (glyoxal-based fixative), Hope (Hepes-glutamic acid buffermediated organic solvent fixative), and/or Zinc Formal-Fixx(Formaldehyde fixative which contains zinc).

In yet another embodiment of the present invention, it is provided, thatthe information of the method disclosed herein is combines with standardhistopathological data, such as TNM status, Grade, Location, Cell Type,Inflammatory status, to improve the validity of the result and/or adoptto the clinical situation.

In yet another embodiment of the present invention, it is provided, thatthe results are adjusted to tumor cell content or sublocalization of thetissue material within the malignant tissue, e.g., invasive front,central oarts, angiogenic subregion, inflammatory region, etc.

Routinely, in tumor diagnosis tissue samples are taken as biopsies froma patient and undergo diagnostic procedures. For this purpose, thesamples are fixed in formaline, embedded in paraffine and are thenexamined with immunohistochemistry methods. The formaline treatmentleads to the inactivation of enzymes, as for example the ubiquitousRNA-digesting enzymes (RNAses). For this reason, the mRNA status of thetissue (the so called transcriptome), remains unaffected.

However, the formaline treatment leads to partial depolymerization ofthe individual mRNA molecules. Same applies for other fixatives, as forexample mentioned in the above enumeration.

For this reason, it is provided in an embodiment of the presentinvention that after lysis, the sample is treated with silica-coatedmagnetic particles and a chaotropic salt, for purification of thenucleic acids contained in said sample for further determination.

However, the isolation method may alternatively also be silica columnbased with or without chaotropic agents.

Collaborators of the inventor of the present invention have developed anapproach which however allows successful purification of mRNA out oftissue samples fixed in such manner, and which is disclosed, amongothers, in WO03058649, WO2006136314A1 and DE10201084A1, the content ofwhich is incorporated herein by reference. Said method comprises the useof magnetic particles coated with silica (SiO₂). The silica layer isclosed and tight and is characterized by having an extremely smallthickness on the scale of a few nanometers. These particles are producedby an improved method that leads to a product having a closed silicalayer and thus entail a highly improved purity. The said method preventsan uncontrolled formation of aggregates and clusters of silicates on themagnetite surface whereby positively influencing the additional citedproperties and biological applications. The said magnetic particlesexhibit an optimized magnetization and suspension behavior as well as avery advantageous run-off behavior from plastic surfaces. These highlypure magnetic particles coated with silicon dioxide are used forisolating nucleic acids, including DNA and RNA, from cell and tissuesamples, the separating out from a sample matrix ensuing by means ofmagnetic fields. These particles are particularly well-suited for theautomatic purification of nucleic acids, mostly from biological bodysamples for the purpose of detecting them with different amplificationmethods.

The selective binding of these nucleic acids to the surface of saidparticles is due to the affinity of negatively charged nucleic acids tosilica containing media in the presence of chaotropic salts likeguanidinisothiocyanate. Said binding properties are known as the socalled “boom principle”. They are described in the European PatentEP819696, the content of which is incorporated herein by reference.

The said approach is particularly useful for the purification of mRNAout of formaline and/or paraffine fixed tissue samples. In contrast tomost other approaches, which leave very small fragments behind that arenot suitable for later determination by PCR and/or hybridizationtechnologies, the said approach creates mRNA fragments which are largeenough to allow specific primer hybridization and/or specific probehybridization. A minimal size of at least about 50 base pairs, or atleast about 100 base pairs, or at least about 200 base pairs is neededfor specific and robust detection of target gene expression. Moreover itis also necessary to not have too many inter-sample variations withregard to the size of the RNA fragments to guarantee comparability ofgene expression results. Other issues of perturbance of expression databy sample preparation problems relate to the contamination level withDNA, which is lower compared to other bead or column based technologies.

The said approach thus allows a highly specific determination of thestatus of hormone receptors selected from the group comprising estrogenreceptor, progesterone receptor and/or androgen receptor with one of theabove introduced methods, particularly with hybridization based methods,PCR based methods and/or array based methods, even in fixed routinetissue samples, and is thus extremely beneficial in the context of thepresent invention, as it allows the use of tissue samples fixed withformaline and/or paraffine, which are available in tissue banks andconnected to clinical databases of sufficient follow-up to allowretrospective analysis. Another important aspect is that the saidapproach allows the simultaneous determination of more than one analyte(multiplexing), and is thus ideally suited for the determination ofhormone receptors selected from the group comprising estrogen receptor,progesterone receptor and/or androgen receptor especially ESR1, ESR2,PGR and/or of one or more housekeeping genes in said sample.Alternatively to housekeeping genes, which are per definition beingexpressed in virtually all cells to similar amounts, tumor specific,endothelial cell specific and or stroma specific genes may be includedto further increase the diagnostic precision of said method. By thisapproach one can derive a calibration factor in order to normalize theexpression values of the target genes in samples which have differentshares of tumor tissue and nontumor tissue.

In yet another embodiment of the present invention, it is provided thatsaid endocrine treatment is a hormonal treatment and/or antihormonaltreatment.

Said endocrine treatment may comprises the administration of antagonistsof estrogen binding to the estrogen receptor, estrogen reuptakeinhibitors, selective estrogen receptor downregulators, or as inhibitorsof estrogen biosynthesis, such as aromatase inhibitors. Said endocrinetreatment may also comprise similar approaches to target progesteroneand/or androgen receptors.

In yet another embodiment of the invention a method for correlating theclinical outcome of a patient suffering from or at risk of developing alung cancer with the presence or non-presence of a defect in expressionlevels of the RNA transcripts of at least one gene encoding for ahormone receptor selected from the group comprising estrogen receptor,progesterone receptor and/or androgen receptor is provided, said methodcomprising the steps of:

-   -   a. obtaining a fixed biological sample from said patient;    -   b. determining the expression levels of the RNA transcripts of        at least one gene encoding for a hormone receptor selected from        the group comprising estrogen receptor, progesterone receptor        and/or androgen receptor, and    -   c. correlating the pattern of expression level(s) determined        in (b) with said patient's data, said data being selected from        the group consisting of etiopathology data, clinical symptoms,        anamnesis data and/or data concerning the therapeutic regimen.

The said method is particularly beneficial for epidemiological studies.These studies profit from the fact that large tissue databases existcomprising paraffin and/or formalin fixed tissue samples together withan extensive documentation of the patient's history, includingetiopathology data, clinical symptoms, anamnesis data and/or dataconcerning the therapeutic regimen. The said methods advantageouslyallows for large scale studies.

In another embodiment of the present invention, a kit useful forcarrying out a method of the invention, comprising at least a pair ofgene specific primers and/or probes each having a sequence sufficientlycomplementary to at least one gene or gene fragments or genomic nucleicacid sequence encoding for a at least one gene coding for a hormonereceptor selected from the group comprising estrogen receptor,progesterone receptor and/or androgen receptor for quantifying theexpression of said at least one gene or gene fragment or genomic nucleicacid sequence, and/or their fractions, variants, homologues,derivatives, fragments, complements, hybridizing counterparts, ormolecules sharing a sequence identity of at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, or at least about 97%.

These nucleic acids can be used either as primers for a polymerase chainreaction protocol, or as detectable probes for monitoring the saidprocess.

Furthermore, it is provided that the said nucleic acid or nucleic acidhomologue is selected from the group consisting of DNA, RNA, PNA, LNAand/or morpholino. The nucleic acid may, in a given embodiment, belabeled with at least one detectable marker. This feature is applicableparticularly for those nucleic acids which serve as detectable probesfor monitoring the polymerase chain reaction process.

Such detectable markers may for example comprise at least one labelselected from the group consisting of fluorescent molecules, luminescentmolecules, radioactive molecules, enzymatic molecules and/or quenchingmolecules.

In another embodiment, the said detectable probes are labeled with afluorescent marker at one end and a quencher of fluorescence at theopposite end of the probe. The close proximity of the reporter to thequencher prevents detection of its fluorescence; breakdown of the probeby the 5′ to 3′ exonuclease activity of the taq polymerase breaks thereporter-quencher proximity and thus allows unquenched emission offluorescence, which can be detected. An increase in the product targetedby the reporter probe at each PCR cycle therefore causes a proportionalincrease in fluorescence due to the breakdown of the probe and releaseof the reporter.

The oligonucleotide in one embodiment of the invention comprises anucleotide sequence which is a fragment, a fraction, a variant, ahomologue, a derivative of, or a complementary to, any of the nucleicacid molecules set forth as SEQ ID NOs 1-9, or which is capable ofhybridizing to a fragment, a fraction, a variant, a homologue, or aderivative of any of the nucleic acid molecules set forth as SEQ ID NOs1-9.

DISCLAIMER

To provide a comprehensive disclosure without unduly lengthening thespecification, the applicant hereby incorporates by reference each ofthe patents and patent applications referenced above.

The particular combinations of elements and features in theabove-detailed embodiments are exemplary only; the interchanging andsubstitution of these teachings with other teachings in this and thepatents/applications incorporated by reference are also expresslycontemplated. As those skilled in the art will recognize, variations,modifications, and other implementations of what is described herein canoccur to those of ordinary skill in the art without departing from thespirit and the scope of the invention as claimed. Accordingly, theforegoing description is by way of example only and is not intended aslimiting. The invention's scope is defined in the following claims andthe equivalents thereto. Furthermore, reference signs used in thedescription and claims do not limit the scope of the invention asclaimed.

TABLE 1 Genes of interest Gene_Symbol Ref. Sequences Ref. SequencesUnigene_ID [A] Description [A] [A] [A] ESR1 Estrogen receptor NM000125.2 Hs.208124 ESR2 Estrogen receptor NM 001040276+1 Hs.525392;HS660607 PGR Progesterone NM 000926.4 Hs.368072 receptor AR Androgenreceptor NM 000044+2 Hs.496240 AR Androgen receptor NM 001011645+1Hs.496240 CYP19 Aromatase NM 000103.3 miRNA 205 miRNA 221 miRNA 222SNAI1 Snail homolog 1 NM 005985.2 Hs.48029 SNAI2 SLUG; Snail NM 003068.3Hs.360174 homolog 2 SNAI3 SMUC; Snail NM 17810.3 Hs.673548 homolog 3

The terms “Ref. Sequences” and “Unigene ID” relate to databases in whichthe respective proteins are listed under the given access number. Thesedatabases can be accessed over the NCBI server.

Additional details, features, characteristics and advantages of theinvention are disclosed in the following examples that, in an exemplaryfashion, show embodiments of the present invention. However, theseexamples should by no means be understood as to limit the scope of theinvention.

Example 1 Measurement of ESR1 by RT PCR

Molecular Methods

RNA was isolated from formalin-fixed paraffin-embedded (“FFPE”) tumortissue samples employing an experimental method based on proprietarymagnetic beads from Siemens Medical Solutions Diagnostics. In short, theFFPE slide were lysed and treated with Proteinase K for 2 hours 55° C.with shaking After adding a binding buffer and the magnetic particles(Siemens Medical Solutions Diagnostic GmbH, Leverkusen, Germany) nucleicacids were bound to the particles within 15 minutes at room temperature.On a magnetic stand the supernatant was taken away and beads were washedseveral times with washing buffer. After adding elution buffer andincubating for 10 min at 70° C. the supernatant was taken away on amagnetic stand without touching the beads. After normal DNAse Itreatment for 30 minutes at 37° C. and inactivation of DNAse I thesolution was used for reverse transcription-polymerase chain reaction(RT-PCR).

RT-PCR was run as standard kinetic one-step Reverse TranscriptaseTaqMan™ polymerase chain reaction (RT-PCR) analysis on a ABI7900(Applied Biosystems) PCR system for assessment of mRNA expression. Rawdata of the RT-PCR were normalized to one or combinations of thehousekeeping genes RPL37A, GAPDH, RPL13, and HPRT1 by using thecomparative ΔΔCT method, known to those skilled in the art. In brief, atotal of 40 cycles of RNA amplification were applied and the cyclethreshold (CT) of the target genes was set as being 0.5. CT scores werenormalized by subtracting the CT score of the housekeeping gene RPL37Aor the mean of the combinations from the CT score of the target gene(Delta CT). RNA results were then reported as 40-Delta CT or2^(((40-(CT Target Gene-CT Housekeeping Gene)*(−1))))(2̂(40−(CT TargetGene−CT Housekeeping Gene)*(−1))) scores, which would correlateproportionally to the mRNA expression level of the target gene. For eachgene specific Primer/Probe were designed by Primer Express® softwarev2.0 (Applied Biosystems) according to manufactures instructions.

Statistics

The statistical analysis was performed with Graph Pad Prism Version 4(Graph Pad Prism Software, Inc).

The clinical and biological variables were categorized into normal andpathological values according to standard norms. The Chi-square test wasused to compare different groups for categorical variables. To examinecorrelations between different molecular factors, the Spearman rankcorrelation coefficient test was used.

For univariate analysis, logistic regression models with one covariatewere used when looking at categorical outcomes. Survival curves wereestimated by the method of Kaplan and Meier, and the curves werecompared according to one factor by the log rank test. For theestimation of multivariate models, all parameters which were significantat the univariate analysis (p<0.05) were fitted to a Cox regressionmodel using a backward forward stepwise method for the selection ofcovariates. Confidence intervals (CI) at 95% for hazard rates (HR) werecalculated. All the probabilities that were calculated were two-tailed.

Experiments have repeatedly shown that determination of hormone receptorstatus by RT PCR consistently yielded better results than analysis byimmunohistochemistry (IHC), i.e., while no stratification of patientscould be achieved by analysis of IHC data, analysis of gene expressiondata obtained by PCR based methods consistently yielded significantresults allowing a reliable stratification of patients in to high riskand low risk groups.

Example 2 Determination of ESR1 Expression Using RT-PCR in a Lung CancerPatient Cohort

Hormone receptor RNA expression of ESR1, ESR2, PGR, AR was analyzed byAffymetrix array technologies and kPCR technologies by employing astandardized RNA-extraction method based on proprietary magnetic beadsfrom Siemens Healthcare Diagnostics and using standard Taqman® PCRMethodology on the ABI7900 PCR system. Fresh tissue biopsy samples andtumor resectates of stage III and IV small cell and non small cell lungcancer patients kPCR from a non-stratified, population based cohorttreated with chemotherapy (n=83) were analyzed.

By correlation analysis, it was surprisingly found that overexpressionof ESR1 as assessed by Affymetrix and kPCR analysis indicates goodprognosis of lung cancer patients treated by standard chemotherapy asindicated by prolonged disease free and overall survival. ESR1expression displayed a broad range of relative copy number (2.5 logs) asdetermined by standard kPCR technologies after normalization to varioushousekeeping genes (RPL37A, GAPDH, RPL13, HPRT1, CALM2) in thepopulations based cohort (n=83) of patients with both SCLC and NSCLC. Bytaking technical cut-offs like the median and tertiles, it was shownthat high expression is related to prolonged survival. Importantly, theESR1 and AR expressing tumor group may benefit most from endocrinetreatment options. The test could be used for stratification of lungcancer patients towards endocrine treatments in the late and alsoearlier setting. As the median expression of ESR1 and AR was lower thanin breast cancer and there is need for quantitative assessment of ESR1and AR expression to reliably select patients, it is reasonable toexpect a technical superiority of the present approach over standardtechnologies (i.e., immunohistochemistry) will also persist also in lungcancer.

Example 3 Determination of ESR Expression Using RT-PCR in NSCLC PatientCohort

By correlation analysis, it was surprisingly found that overexpressionof ESR1 as kPCR analysis indicates good prognosis of non small cell lungcancer (NSCLC) patients (male and female Caucasian patients) treated bystandard chemotherapy as indicated by prolonged disease free and overallsurvival. ESR1 expression displayed a broad range of relative copynumber (2.5 logs) as determined by standard kPCR technologies afternormalization to various housekeeping genes (RPL37A, GAPDH, RPL13,HPRT1, CALM2) in the populations based cohort (n=35) of patients withNSCLC. By taking technical cut-offs like the median and tertiles, it wasshown that high expression is related to prolonged survival. Results forESR1 expression greater or lower than median are shown in FIG. 1.Patients stratified according to ESR1 expression above or below thethird quartile of ESR1 expression are shown in FIG. 2. The majority ofpatients were treated with a platinum-based regimen. Samples were FFPEtissue analyzed with RT-kPCR. The median follow up 9 month; 82% StageIV; patient number was n=35.

Example 4 Correlation of ESR1 Expression Determined by Using RT-PCR withSite of Initial Metastasis in NSCLC Patient Cohort

By correlation analysis, it was surprisingly found that high ESR1expression positively correlates with development of bone metastasisalso in NSCLC patients as depicted in FIG. 3. In addition, metastasis tothe adrenal gland trends to be significantly associated with adrenalmetastasis, which is in females the major source of androgens. Tropismof hormone receptor positive NSCLC cells towards the endocrine gland hasnot been described before but is in line with the surprising findingthat a subtype of NSCLC cells derives growth advantage from hormonessuch as androgens or its derivatives (estrogen).

Example 5 Spearman Correlation of ESR1, SNAI2, CDH1, CDH11 ExpressionDetermined by Using RT-PCR with Site of Initial Metastasis in NSCLCPatient Cohort

In view of the extreme effect of hormone receptors on patient survivalin NSCLC, the inventor has analyzed the promoter sites regulating theexpression of hormone receptor RNA expression, i.e., ESR1, ESR2, PGR, ARto identify candidate genes that oppose the effect of ESR1 andcontribute to more aggressive and hormone insensitive tumor subtypes.Thereby he had the idea to first analyze the isoform specific expressionof ESR1 in cancer and then analyze the respective isoform specificpromoter sites in molecular detail. Surprisingly, he has identifiedtumor specific ESR1 isoforms, whose promoter region in turn exhibitedseveral snail transcription factor binding sites (i.e., bindings sitesfor SNAI1, SNAI2 and SNAI3). The interaction of hormone receptors andsnail factors was then analyzed in diverse Affymetrix data sets. As oneexample, fresh tissue biopsy samples (laparoscopy; pre-treatment) andtumor resectates (surgery; post-treatment) of stage III and IV ovariancancer patients (n=40) neoadjuvantly treated with chemotherapy (6×AUC)were analyzed by Affymetrix expression profiling. By Spearmancorrelation analysis and as depicted in FIG. 4, it could be proven thatthe transcription factor SNAI2 indeed strongly and negatively correlatedwith ESR1 (r=−0.56; p=0.0004) followed by E-Cadherin (“CDH1”; r=−0.36;p=0.03), both of which are associated with epithelial and good prognosisphenotype. Conversely, SNAI2 positively correlated with MMP2 (r=0.86;p<0.0001), Spon2 (r=0.80; p<0.0001), ADAM12 (r=0.72; p<0.0001) andOB-Cadherin (“CDH11”; r=0.66; p=0.03). Surprisingly the dramatic switchof cell-cell-adhesion from E-Cadherin to OB-Cadherin further illustratedthe Epithelial-Mesenchymal transition, which might be associated withhighly invasive behavior of tumor cells. However, the presence ofOB-Cadherin might also indicate successful recruitment ofosteoblast-like bone marrow cells into the primary tumor site furtherindicating the more aggressive phenotype of ESR1 low expressing andsimultaneously SNAI2 overexpressing tumors.

Example 6 Determination of ESR1 in Combination with SNAI2 ExpressionUsing Array Profiling in Ovarian Cancer Patient Cohort

A two gene-ratio was generated by dividing SNAI2 by ESR1. As depicted inFIG. 5, Kaplan-Meier-Analysis revealed that ovarian cancer patientshaving high two-gene-ratio values (Cut-Off 0.21), indicating high SNAI2expression and simultaneously low ESR1 expression, which accounts forapproximately one third of the ovarian cancer patients, have a worseoverall survival than patients having high ESR1 expression and low SNAI2expression. The latter exhibited 100% overall survival at three yearswithin this stage IV neoadjuvantly treated ovarian cancer cohort (Hazardratio 0.00; p=0.0021; Median Recurrence free Survival not reached versus24, 8 months; 100% Overall Survival versus 20% Overall Survival at threeyears of follow-up). By generating a two gene ratio of SNAI2 and ESR1the test can be performed without using any housekeeping gene, whichfurther limits the number of required genes and reduces complexity andcosts for performing the assay. The test could be used forstratification of cancer patients towards targeted treatments in thelate and also earlier setting. Particularly SNAI2 negatively correlateswith PARP1 (r=−0.54; p=0.0004) meaning that high expression of SNAI2 isassociated with low expression of PARP 1. PARP1 is the target of PARPInhibitors. Lower expression of PARP1 (and/or BRCA1) indicatesresponsiveness towards this regimen. However, the expression of PARP1 isvery difficult to determine on protein and mRNA level, due to comparablylow general expression and low dynamic range. However patientsresponding to PARP Inhibitors could be more easily detected bydetermining the balance between hormone receptors and SNAI factors.

Example 7 Determination of ESR1 in Combination with SNAI2 ExpressionUsing Array Profiling in Non-Small Cell Lung Cancer Patient Cohort

To further validate, that the two gene-ratio generated by dividing SNAI2by ESR1 is also prognostic in other cancer indications and particularlyin lung cancer, the public available whole genome Affymetrix geneexpression data from Jinkook Kim (GSE8894; Lee E S et al. (2008):Prediction of Recurrence-free survival in postoperative non-small celllung cancer patients by using an integrated model of clinical and geneexpression. Clin Cancer Res. 14(22): 7397-404) was retrieved from theGEO database. In brief, a total of 253 fresh frozen non-small lungcancer tumor samples from patients who underwent curative resection ofNSCLC at Samsung Medical Center in Seoul (South Korea) between January1995 and December 2005 were selected and acceptable RNA quality formicroarray analysis was achieved from 138 tumors.

The first validation focused on female NSCLC patients (n=34;Adenocarcinoma and Squamous Cell Carcinoma). The two-gene ratio wasconstructed by dividing SNAI2 by ESR1 expression values. According tothis invention low gene-ratio values reflect lower expression of ESR1and simultaneously higher expression of ESR1 and are associated withlower risk of recurrence. By using a two gene-ratio cut-off at 2.32 alow risk was predicted for approximately 30% of the women (i.e., 11/34patients). As depicted in FIG. 6, the Kaplan-Meier analysis validatedthat the low-risk prediction by using the SNAI2 and ESR1 expressionratio have a lower risk of recurrence (Hazard ratio 0.25; p=0.0012;Median Recurrence free Survival not reached versus 9.8 months; 70%Recurrence Free Survival versus 20% Recurrence Free Survival at twoyears of follow-up). This validates the prognostic significance of SNAI2and ESR1 in independent patients. Importantly, this indicates that theinvention not only works for metastatic, Caucasian NSCLC patientstreated within a first-line chemotherapy protocol as depicted in Example3, but also works in non-metastatic, Asian NSCLC patients after curativeresection of the tumor mass. This means, that the invention is suitablefor NSCLC patients at different disease and treatment stages.Importantly, as estrogen receptor is capable of triggering tumor growth,the determination of hormone receptors (particularly ESR1) and/or Snailfactors (particularly SNAI2) is important for peri- or post-menopausalwomen, to evaluate possible risks associated with hormone replacementtherapy, as treatment with hormones could force tumor growth andaggressiveness particularly in ESR1 high expressors and/or SNAI2 lowexpressors.

Example 8 Determination of PGR Using Array Profiling in Non-Small CellLung Cancer Patient Cohort

The second validation focused on male NSCLC patients (n=100;Adenocarcinoma and Squamous Cell Carcinoma). As depicted in FIG. 7 theKaplan-Meier analysis validated that high PGR expression indicates lowerrisk of recurrence (Hazard ratio 0.46; p=0.0056; Median Recurrence freeSurvival not reached versus 23 months; 65% Recurrence Free Survivalversus 40% Recurrence Free Survival at three years of follow-up). Thisvalidates that hormone receptor expression is significant also for maleNSCLC in Asian patients having undergone curative resection of theprimary tumor (see above).

What is claimed is:
 1. A method of classifying a sample of a patient whosuffers from or being at risk of developing cancer, said methodcomprising the steps of: a. determining in said sample from saidpatient, on a non protein basis, the expression level of at least onegene encoding for a hormone receptor selected from the group comprisingestrogen receptor, progesterone receptor and/or androgen receptor insaid sample; b. comparing the one or more expression level(s) determinedin step (a) with one or more expression level(s) of one or morereference genes; and c. classifying the sample of said patient from theoutcome of the comparison in step (b) into one of at least twoclassifications.
 2. The method according to claim 1, wherein a mode oftreatment based on the classification in step (c) comprises a treatmenttargeting at least one hormone receptor selected from the groupcomprising estrogen receptor, progesterone receptor and/or androgenreceptor, or targeting their respective signaling pathways, and/or atreatment targeting repair mechanisms related therewith.
 3. The methodaccording to claim 1 or 2, characterized in that said treatmentcomprises the administration of tamoxifen.
 4. The method according toany of the aforementioned numbered paragraphs, characterized in thatsaid treatment is intended to be given as hormone replacement therapy(HRT) in peri- or postmenopausal women.
 5. The method according to claimany of the aforementioned claims, characterized in that the geneencoding for the estrogen receptor is ESR1.
 6. A method according any ofthe aforementioned claims, characterized in that said one or morereference gene(s) is at least one housekeeping gene and/or at least oneEMT marker gene.
 7. The method according to claim 6, wherein the atleast one housekeeping gene is selected from the group comprisingRPL37A, GAPDH, RPL 13 and/or HPRT1; and the at least one EMT marker geneis selected from the group comprising SNAI1, SNAI2 and/or SNAI3.
 8. Themethod according to any of the aforementioned claims, wherein thecomparing step (b) is a ratio between the expression level of at leastone hormone receptor and at least one EMT marker gene.
 9. The methodaccording to any of the aforementioned claims, wherein the comparingstep (b) is a ratio of ESR1 to SNAI2.
 10. The method according to anyone of the aforementioned claims, wherein said expression level(s) isdetermined by a. a hybridization based method; b. a PCR based method; c.a method based on the electrochemical detection of particular molecules,and/or d. an array based method.
 11. The method according to any one ofthe aforementioned claims, characterized in that said expression levelis determined by reverse transcriptase polymerase chain reaction of RNAtranscripts.
 12. The method according to claim 10, characterized in thatsaid expression level is determined in formalin and/or paraffin fixedtissue samples of the RNA transcripts.
 13. The method according to anyone of the aforementioned claims, wherein, after lysis, the sample istreated with silica-coated magnetic particles and a chaotropic salt, forpurification of the nucleic acids contained in said sample prior to thedetermination in step (a).
 14. The method according to any one of theaforementioned claims, characterized in that said cancer displayscharacteristics of, or is, an adenocarcinoma.
 15. The method accordingto any one of the aforementioned claims, characterized in that saidcancer is selected from the group comprising lung cancer, a non-smallcell lung cancer (NSCLC), ovarian cancer; breast cancer and/or prostatecancer.
 16. The method according to any one of the aforementionedclaims, wherein the expression level(s) determined in step (a) is/arecorrelated with said patient's data, said data being selected from thegroup consisting of etiopathology data, clinical symptoms, anamnesisdata and/or data concerning the therapeutic regimen.
 17. Anoligonucleotide comprising a nucleotide sequence which is a fragment, afraction, a variant, a homologue, a derivative of, or a complementaryto, any of the nucleic acid molecules set forth as SEQ ID NOs 1-9, orwhich is capable of hybridizing to a fragment, a fraction, a variant, ahomologue, or a derivative of any of the nucleic acid molecules setforth as SEQ ID NOs 1-9.
 18. The oligonucleotide according to claim 17,wherein said oligonucleotide is selected from the group consisting of a.an amplification primer b. a labeled probe, and/or c. a substrate boundprobe.
 19. A kit useful for carrying out a method of any one of theaforementioned claims, comprising at least one oligonucleotide accordingto claim 17 and/or 18.